Stable subcutaneous protein formulations and uses thereof

ABSTRACT

The present invention relates generally to stable formulations comprising CTLA4Ig molecules, including lyophilized, and liquid formulations for administration via various routes including, for example, routes such as intravenous (IV) and subcutaneous (SC) for treating immune system diseases and tolerance induction.

The present patent application is a continuation of U.S. Ser. No.13/796,586 filed Mar. 12, 2013, now allowed, which is a divisionalapplication of U.S. Ser. No. 12/086,876 filed Jun. 4, 2009, issued asU.S. Pat. No. 8,476,239, which is a 371 of PCT/US2006/062297 filed Dec.19, 2006, which claims the priority of provisional application U.S. Ser.No. 60/752,150, filed on Dec. 20, 2005, the contents of which are herebyincorporated by reference in their entirety into this application

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

FIELD OF THE INVENTION

The present invention relates generally to stable formulationscomprising CTLA4Ig molecules, including lyophilized, and liquidformulations for administration via various routes including, forexample, routes such as intravenous (IV) and subcutaneous (SC).

BACKGROUND OF THE INVENTION

Over the past two decades, recombinant DNA technology has led to thecommercialization of many protein therapeutics. The most conventionalroute of delivery for protein drugs has been intravenous (IV)administration because of poor bioavailability by most other routes,greater control during clinical administration, and fasterpharmaceutical development. For products that require frequent andchronic administration, the alternate subcutaneous (SC) route ofdelivery is more appealing. When coupled with pre-filled syringe andautoinjector device technology, SC delivery allows for homeadministration and improved compliance of administration.

Treatments with high doses of more than 1 mg/kg or 100 mg per dose oftenrequire development of formulations at concentrations exceeding 100mg/ml because of the small volume (<1.5 ml) that can be given by the SCroutes. For proteins that have a propensity to aggregate at the higherconcentrations, achieving such high concentration formulations is adevelopmental challenge. Even for the IV delivery route, where largevolumes can be administered, protein concentrations of tens ofmilligrams per milliliter may be needed for high dosing regimens andthis may pose stability challenges for some proteins.

The principles governing protein solubility are more complicated thanthose for small synthetic molecules, and thus overcoming the proteinsolubility issue takes different strategies. Operationally, solubilityfor proteins could be described by the maximum amount of protein in thepresence of co-solutes whereby the solution remains visibly clear (i.e.,does not show protein precipitates, crystals, or gels). The dependenceof protein solubility on ionic strength, salt form, pH, temperature, andcertain excipients has been mechanistically explained by changes in bulkwater surface tension and protein binding to water and ions versusself-association by Arakawa et al in Theory of protein solubility,Methods of Enzymology, 114:49-77, 1985; Schein in Solubility as afunction of protein structure and solvent components, BioTechnology8(4):308-317, 1990; Jenkins in Three solutions of the protein solubilityproblem, Protein Science 7(2):376-382, 1998; and others. Binding ofproteins to specific excipients or salts influences solubility throughchanges in protein conformation or masking of certain amino acidsinvolved in self-interaction. Proteins are also preferentially hydrated(and stabilized as more compact conformations) by certain salts, aminoacids, and sugars, leading to their altered solubility.

Aggregation which requires bi-molecular collision is expected to be theprimary degradation pathway in protein solutions. The relationship ofconcentration to aggregate formation depends on the size of aggregatesas well as the mechanism of association. Protein aggregation may resultin covalent (e.g., disulfide-linked) or non-covalent (reversible orirreversible) association. Irreversible aggregation by non-covalentassociation generally occurs via hydrophobic regions exposed by thermal,mechanical, or chemical processes that alter a protein's nativeconformation. Protein aggregation may impact protein activity,pharmacokinetics and safety, e.g., due to immunogenicity.

A typical approach to minimize aggregation, is to restrict the mobilityof proteins in order to reduce the number of collisions. Lyophilizationwith appropriate excipients may improve protein stability againstaggregation by decreasing protein mobility and by restrictingconformational flexibility with the added benefit of minimizinghydrolytic reactions consequent to removal of water. The addition ofappropriate excipients, including lyoprotectants, can prevent theformation of aggregates during the lyophilization process as well asduring storage of the final product. A key parameter for effectiveprotection is the molar ratio of the lyoprotectant to the protein.Generally molar ratios of 300:1 or greater are required to providesuitable stability, especially for room temperature storage. Such ratioscan also, however, lead to an undesirable increase in viscosity.

Lyophilization allows for designing a formulation with appropriatestability and tonicity. Although isotonicity is not necessarily requiredfor SC administration, it may be desirable for minimizing pain uponadministration. Isotonicity of a lyophile is difficult to achievebecause both the protein and the excipients are concentrated during thereconstitution process. Excipient:protein molar ratios of 500:1 willresult in hypertonic preparations if the final protein concentration istargeted for >100 mg/ml. If the desire is to achieve an isotonicformulation, then a choice of lower molar ratio of excipient:proteinwill result in a potentially less stable formulation.

Determining the highest protein concentration achievable remains anempirical exercise due to the labile nature of protein conformation andthe propensity to interact with itself, with surfaces, and with specificsolutes.

Examples of subjects who may benefit from SC formulations are those thathave conditions that require frequent and chronic administration such assubjects with the immune system disease rheumatoid arthritis and immunedisorders associated with graft transplantation. Commercially availableprotein drug products for the treatment of rheumatoid arthritis includeHUMIRA®, ENBREL® and REMICADE®.

HUMIRA® (Abbott) is supplied in single-use, 1 ml pre-filled glasssyringes as a sterile, preservative-free solution for subcutaneousadministration. The solution of HUMIRA® is clear and colorless, with apH of about 5.2. Each syringe delivers 0.8 ml (40 mg) of drug product.Each 0.8 ml of HUMIRA® contains 40 mg adalimumab, 4.93 mg sodiumchloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mg dibasicsodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 mg citric acidmonohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80 and Water forInjection, USP. Sodium hydroxide added as necessary to adjust pH.

ENBREL® (Amgen) is supplied in a single-use pre-filled 1 ml syringe as asterile, preservative-free solution for subcutaneous injection. Thesolution of ENBREL® is clear and colorless and is formulated at pH6.3±0.2. Each ENBREL® single-use prefilled syringe contains 0.98 ml of a50 mg/ml solution of etanercept with 10 mg/ml sucrose, 5.8 mg/ml sodiumchloride, 5.3 mg/ml L-arginine hydrochloride, 2.6 mg/ml sodium phosphatemonobasic monohydrate and 0.9 mg/ml sodium phosphate dibasic anhydrous.Administration of one 50 mg/ml prefilled syringe of ENBREL® provides adose equivalent to two 25 mg vials of lyophilized ENBREL®, when vialsare reconstituted and administered as recommended. ENBREL® multiple-usevial contains sterile, white, preservative-free, lyophilized powder.Reconstitution with 1 ml of the supplied Sterile Bacteriostatic Waterfor Injection (BWFI), USP (containing 0.9% benzyl alcohol) yields amultiple-use, clear, and colorless solution with a pH of 7.4±0.3containing 25 mg etanercept, 40 mg mannitol, 10 mg sucrose, and 1.2 mgtromethamine.

REMICADE® (Centocor) is supplied as a sterile, white, lyophilized powderfor intravenous infusion. Following reconstitution with 10 ml of SterileWater for Injection, USP, the resulting pH is approximately 7.2. Eachsingle-use vial contains 100 mg infliximab, 500 mg sucrose, 0.5 mgpolysorbate 80, 2.2 mg monobasic sodium phosphate, monohydrate, and 6.1mg dibasic sodium phosphate, dihydrate. No preservatives are present.

Commercially available protein drug products for the treatment of immunedisorders associated with graft transplantation include SIMULECT®, andZENAPAX®.

The drug product, SIMULECT® (Novartis), is a sterile lyophilisate whichis available in 6 ml colorless glass vials and is available in 10 mg and20 mg strengths. Each 10-mg vial contains 10 mg basiliximab, 3.61 mgmonobasic potassium phosphate, 0.50 mg disodium hydrogen phosphate(anhydrous), 0.80 mg sodium chloride, 10 mg sucrose, 40 mg mannitol and20 mg glycine, to be reconstituted in 2.5 ml of Sterile Water forInjection, USP. Each 20-mg vial contains 20 mg basiliximab, 7.21 mgmonobasic potassium phosphate, 0.99 mg disodium hydrogen phosphate(anhydrous), 1.61 mg sodium chloride, 20 mg sucrose, 80 mg mannitol and40 mg glycine, to be reconstituted in 5 ml of Sterile Water forInjection, USP. No preservatives are added.

ZENAPAX® (Roche Laboratories), 25 mg/5 ml, is supplied as a clear,sterile, colorless concentrate for further dilution and intravenousadministration. Each milliliter of ZENAPAX® contains 5 mg of daclizumaband 3.6 mg sodium phosphate monobasic monohydrate, 11 mg sodiumphosphate dibasic heptahydrate, 4.6 mg sodium chloride, 0.2 mgpolysorbate 80, and may contain hydrochloric acid or sodium hydroxide toadjust the pH to 6.9. No preservatives are added

CTLA4Ig molecules interfere with T cell costimulation by inhibiting theCD28-B7 interaction. Therefore, CTLA4Ig molecules can provide atherapeutic use for immune system diseases, such as rheumatoid arthritisand immune disorders associated with graft transplantation.

There is a need for a stable, effective convenient formulationscomprising CTLA4Ig molecules for treatment of immune system disorders.

SUMMARY OF THE INVENTION

The present invention provides formulations for treating immune systemdiseases, by administering to a subject CTLA4Ig molecules, which bind toB7 molecules on B7-positive cells, thereby inhibiting endogenous B7molecules from binding CTLA4 and/or CD28 on T-cells.

The lyophilized formulation of the invention comprises the CTLA4Igmolecule in a weight ratio of at least 1:2 protein to lyoprotectant. Thelyoprotectant is preferably sugar, more preferably disaccharides, mostpreferably sucrose or maltose. The lyophilized formulation may alsocomprise one or more of the components selected from the list consistingof buffering agents, surfactants, bulking agents and preservatives.

In certain embodiments, the amount of sucrose or maltose useful forstabilization of the lyophilized drug product is in a weight ratio of atleast 1:2 protein to sucrose or maltose, preferably in a weight ratio offrom 1:2 to 1:5 protein to sucrose or maltose, more preferably in aweight ratio of about 1:2 protein to maltose or sucrose.

The subcutaneous (SC) formulation of the invention comprises the CTLA4Igmolecule at a protein concentration of at least 100 mg/ml in combinationwith a sugar at stabilizing levels, preferably a protein concentrationof at least 125 mg/ml in combination with a sugar at stabilizing levels,in an aqueous carrier. The sugar is preferably in a weight ratio of atleast 1:1.1 protein to sugar. The stabilizer is preferably employed inan amount no greater than that which may result in a viscosityundesirable or unsuitable for administration via SC syringe. The sugaris preferably disaccharides, most preferably sucrose. The SC formulationmay also comprise one or more of the components selected from the listconsisting of buffering agents, surfactants, and preservatives.

In certain embodiments, the amount of sucrose useful for stabilizationof the CTLA4Ig molecule SC drug product is in a weight ratio of at least1:1 protein to sucrose, preferably in a weight ratio of from 1:1.3 to1:5 protein to sucrose, more preferably in a weight ratio of about 1:1.4protein to sucrose.

The liquid formulation of the invention comprises the CTLA4Ig moleculeat a protein concentration of at least 20 mg/ml in combination with asugar at stabilizing levels, preferably at least 25 mg/ml in combinationwith a sugar at stabilizing level in an aqueous carrier. Preferably thesugar is in a weight ratio of at least 1:1 protein to sugar. The sugaris preferably disaccharides, most preferably sucrose. The liquidformulation may also comprise one or more of the components selectedfrom the list consisting of buffering agents, surfactants, andpreservatives.

In certain embodiments, the amount of sucrose useful for stabilizationof the liquid drug product is in a weight ratio of at least 1:1 proteinto sucrose, preferably in a weight ratio of from 1:2 to 1:10 protein tosucrose, more preferably in a weight ratio of about 1:2 protein tosucrose.

In another embodiment of the invention, an article of manufacture isprovided which contains the drug product and preferably providesinstructions for its use.

The present invention further provides methods for treating immunesystem diseases and tolerance induction by administering the CTLA4Igmolecule formulations of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the nucleotide sequence (SEQ ID NO:1) of a portion of anexpression cassette for a CTLA4-Ig molecule. Also shown is the aminoacid sequence (SEQ ID NO:2) encoded by the nucleic acid. CTLA4-Igmolecules that can be produced from this expression cassette includemolecules having the amino acid sequence of residues: (i) 26-383 of SEQID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, or(iv) 26-382 of SEQ ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or(vi) 25-383 of SEQ ID NO:2. The expression cassette comprises thefollowing regions: (a) an Oncostatin M signal sequence (nucleotides11-88 of SEQ ID NO: 1; amino acids 1-26 of SEQ ID NO:2); (b) anextracellular domain of human CTLA4 (nucleotides 89-463 of SEQ ID NO:1;amino acids 27-151 of SEQ ID NO:2); (c) a modified portion of the humanIgG1 constant region (nucleotides 464-1159 of SEQ ID NO: 1; amino acids152-383 of SEQ ID NO:2), including a modified hinge region (nucleotides464-508 of SEQ ID NO:1; amino acids 152-166 of SEQ ID NO:2), a modifiedhuman IgG1 CH2 domain (nucleotides 509-838 of SEQ ID NO:1; amino acids167-276 of SEQ ID NO:2), and a human IgG1 CH3 domain (nucleotides839-1159 of SEQ ID NO:1; amino acids 277-383 of SEQ ID NO:2).

FIG. 2 depicts a nucleotide (SEQ ID NO:3) and amino acid (SEQ ID NO:4)sequence of CTLA4-L104EA29Y-Ig (also know as “L104EA29YIg” and “LEA29Y”)comprising a signal peptide; a mutated extracellular domain of CTLA4starting at methionine at position +1 and ending at aspartic acid atposition +124, or starting at alanine at position −1 and ending ataspartic acid at position +124; and an Ig region. SEQ ID NO: 3 and 4depict a nucleotide and amino acid sequence, respectively, ofL104EA29YIg comprising a signal peptide; a mutated extracellular domainof CTLA4 starting at methionine at position +27 and ending at asparticacid at position +150, or starting at alanine at position +26 and endingat aspartic acid at position +150; and an Ig region. L104EA29YIg canhave the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:4,(ii) 26-382 of SEQ ID NO:4; (iii) 27-383 of SEQ ID NO:4 or (iv) 27-382of SEQ ID NO:4, or optionally (v) 25-382 of SEQ ID NO:4, or (vi) 25-383of SEQ ID NO:4.

DETAILED DESCRIPTION OF THE INVENTION

As utilized herein:

A “stable” formulation or drug product is one in which the CTLA4Igmolecule therein essentially retains its physical and chemical stabilityand integrity upon storage. Stability of the CTLA4Ig moleculeformulations can be measured at selected temperatures after selectedtime periods. For example, an increase in aggregate formation followinglyophilization and storage is an indicator for instability of alyophilized CTLA4Ig molecule formulation. In addition to aggregateformation, retention of original clarity, color and odor throughoutshelf life are indicators utilized to monitor stability of CTLA4Igmolecule solutions. HMW species are multimers (i.e. tetramers, hexamers,etc), which have a higher molecular weight than CTLA4Ig molecule dimers.Typically a “stable” drug product may be one wherein the increase inaggregation, as measured by an increase in the percentage of highmolecular weight species (% HMW), is less than about 5% and preferablyless than about 3%, when the formulation is stored at 2-8° C. for oneyear. Preferably, the manufactured drug product comprises less thanabout 25% HMW species, preferably less than about 15% HMW species, morepreferably less than about 10% HMW species, most preferred less thanabout 5% HMW species.

The monomer, dimer and HMW species of CTLA4Ig molecule may be separatedby size exclusion chromatography (SEC). SEC separates molecules based onthe molecular size. Separation is achieved by the differential molecularexclusion or inclusion as the molecules migrate along the length of thecolumn. Thus, resolution increases as a function of column length.CTLA4Ig molecule samples may be separated using a 2695 Alliance HPLC(Waters, Milford, Mass.) equipped with TSK Gel® G3000SWXL (300 mm×7.8mm) and TSK Gel® G3000SWXL (40 mm×6.0 mm) columns (Tosoh Bioscience,Montgomery, Pa.) in tandem. Samples at 10 mg/ml (20 μl aliquot) areseparated using a mobile phase consisting of 0.2 M KH2PO₄, 0.9% NaCl, pH6.8, at a flow rate of 1.0 ml/min. Samples are monitored at anabsorbance of 280 nm using Water's 2487 Dual Wavelength detector. Usingthis system, the HMW species has a retention time of 7.5 min±1.0 min.Each peak is integrated for area under the peak. The % HMW speciescalculated by dividing the HMW peak area by the total peak area.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized formulation in an aqueous carrier such that theCTLA4Ig molecule is dissolved in the reconstituted formulation. Thereconstituted formulation is suitable for intravenous administration(IV) to a patient in need thereof.

An “isotonic” formulation is one which has essentially the same osmoticpressure as human blood. Isotonic formulations will generally have anosmotic pressure from about 250 to 350 mOsmol/KgH2O. The term“hypertonic” is used to describe a formulation with an osmotic pressureabove that of human blood. Isotonicity can be measured using a vaporpressure or ice-freezing type osmometer, for example.

The term “buffering agent” refers to one or more components that whenadded to an aqueous solution is able to protect the solution againstvariations in pH when adding acid or alkali, or upon dilution with asolvent. In addition to phosphate buffers, there can be used glycinate,carbonate, citrate buffers and the like, in which case, sodium,potassium or ammonium ions can serve as counterion.

An “acid” is a substance that yields hydrogen ions in aqueous solution.A “pharmaceutically acceptable acid” includes inorganic and organicacids which are non toxic at the concentration and manner in which theyare formulated.

A “base” is a substance that yields hydroxyl ions in aqueous solution.“Pharmaceutically acceptable bases” include inorganic and organic baseswhich are non-toxic at the concentration and manner in which they areformulated.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest, prevents or reduces chemical and/or physical instability ofthe protein upon lyophilization and subsequent storage.

A “preservative” is an agent that reduces bacterial action and may beoptionally added to the formulations herein. The addition of apreservative may, for example, facilitate the production of a multi-use(multiple-dose) formulation. Examples of potential preservatives includeoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chain compounds), andbenzethonium chloride. Other types of preservatives include aromaticalcohols such as phenol, butyl and benzyl alcohol, alkyl parabens suchas methyl or propyl paraben, catechol, resorcinol, cyclohexanol,3pentanol, and m-cresol.

A “surfactant” is a surface active molecule containing both ahydrophobic portion (e.g., alkyl chain) and a hydrophilic portion (e.g.,carboxyl and carboxylate groups). Surfactant may be added to theformulations of the invention. Surfactants suitable for use in theformulations of the present invention include, but are not limited to,polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer188); sorbitan esters and derivatives; Triton; sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetadine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauramidopropyl-cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-,palmidopropyl-, or isostearamidopropylbetaine (e.g., lauroamidopropyl);myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethylene glycol, polypropyl glycol, and copolymersof ethylene and propylene glycol (e.g., Pluronics, PF68 etc.).

A “drug substance” refers to the starting material utilized informulation of the final drug product. Typical CTLA4Ig drug substancecomposition comprises a protein concentration from 20 mg/ml to 60 mg/ml,pH from 7 to 8 and % HMW species of <5%.

A “formulated bulk solution” refers to the final formulation prior tofilling of the container such as the formulated solution prior tofilling the vials for lyophilization, or the formulated solution priorto filling the syringe for SC injection.

A “drug product” refers to the final formulation packaged in a containerwhich may be reconstituted before use, such as with a lyophilized drugproduct; diluted further before use, such as with a liquid drug product;or utilized as is, such as with a SC solution drug product.

The terms “CTLA4-Ig” or “CTLA4-Ig molecule” or “CTLA4Ig molecule” areused interchangeably, and refer to a protein molecule that comprises atleast a polypeptide having a CTLA4 extracellular domain or portionthereof and an immunoglobulin constant region or portion thereof. Theextracellular domain and the immunoglobulin constant region can bewild-type, or mutant or modified, and mammalian, including human ormouse. The polypeptide can further comprise additional protein domains.A CTLA4-Ig molecule can also refer to multimer forms of the polypeptide,such as dimers, tetramers, and hexamers. A CTLA4-Ig molecule also iscapable of binding to CD80 and/or CD86.

The term “B7-1” refers to CD80; the term “B7-2” refers CD86; and theterm “B7” refers to both B7-1 and B7-2 (CD80 and CD86). The term“B7-1-Ig” or “B7-1Ig” refers to CD80-Ig; the term “B7-2-Ig” or “B7-2Ig”refers CD86-Ig.

In one embodiment, “CTLA4Ig” refers to a protein molecule having theamino acid sequence of residues: (i) 26-383 of SEQ ID NO:2, (ii) 26-382of SEQ ID NO:2; (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ IDNO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ IDNO:2. In monomeric form these proteins can be referred to herein as “SEQID NO:2 monomers,” or monomers “having a SEQ ID NO:2 sequence”. TheseSEQ ID NO:2 monomers can dimerize, such that dimer combinations caninclude, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and(iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and(iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv);(iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and(vi); (v) and (v); (v) and (vi); and, (vi) and (vi). These differentdimer combinations can also associate with each other to form tetramerCTLA4Ig molecules. These monomers, dimers, tetramers and other multimerscan be referred to herein as “SEQ ID NO:2 proteins” or proteins “havinga SEQ ID NO:2 sequence”. (DNA encoding CTLA4Ig as shown in SEQ ID NO:2was deposited on May 31, 1991 with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 under theprovisions of the Budapest Treaty, and has been accorded ATCC accessionnumber ATCC 68629; a Chinese Hamster Ovary (CHO) cell line expressingCTLA4Ig as shown in SEQ ID NO:2 was deposited on May 31, 1991 with ATCCidentification number CRL-10762). As utilized herein “Abatacept” refersto SEQ ID NO:2 proteins.

In one embodiment, CTLA4-L104EA29Y-Ig (sometimes known as “LEA29Y” or“L104EA29Y”) is a genetically engineered fusion protein similar instructure to CTAL4-Ig molecule as shown in SEQ ID NO: 1. L104EA29Y-Ighas the functional extracellular binding domain of modified human CTLA4and the Fc domain of human immunoglobulin of the IgG1 class. Two aminoacid modifications, leucine to glutamic acid at position 104 (L104E),which is position 130 of SEQ ID NO:2, and alanine to tyrosine atposition 29 (A29Y), which is position 55 of SEQ ID NO:2, were made inthe B7 binding region of the CTLA4 domain to generate L104EA29Y. SEQ IDNOS: 3 and 4 depict a nucleotide and amino acid sequence, respectively,of L104EA29YIg comprising a signal peptide; a mutated extracellulardomain of CTLA4 starting at methionine at position +27 and ending ataspartic acid at position +150, or starting at alanine at position +26and ending at aspartic acid at position +150; and an Ig region. DNAencoding L104EA29Y-Ig was deposited on Jun. 20, 2000, with the AmericanType Culture Collection (ATCC) under the provisions of the BudapestTreaty. It has been accorded ATCC accession number PTA-2104.L104EA29Y-Ig is further described in U.S. Pat. No. 7,094,874, issued onAug. 22, 2006, and in WO 01/923337 A2, which are incorporated byreference herein in their entireties.

Expression of L104EA29YIg in mammalian cells can result in theproduction of N- and C-terminal variants, such that the proteinsproduced can have the amino acid sequence of residues: (i) 26-383 of SEQID NO:4, (ii) 26-382 of SEQ ID NO:4; (iii) 27-383 of SEQ ID NO:4 or (iv)27-382 of SEQ ID NO:4, or optionally (v) 25-382 of SEQ ID NO:4, or (vi)25-383 of SEQ ID NO:4. In monomeric form these proteins can be referredto herein as “SEQ ID NO:4 monomers,” or monomers “having a SEQ ID NO:4sequence. These proteins can dimerize, such that dimer combinations caninclude, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and(iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and(iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv);(iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and(vi); (v) and (v); (v) and (vi); and, (vi) and (vi).” These differentdimer combinations can also associate with each other to form tetramerL104EA29YIg molecules. These monomers, dimers, tetramers and othermultimers can be referred to herein as “SEQ ID NO:4 proteins” orproteins “having a SEQ ID NO:4 sequence”. As utilized herein“Belatacept” refers to SEQ ID NO:4 proteins.

CTLA4-Ig Monomers and Multimers

CTLA4-Ig molecules can include, for example, CTLA4-Ig proteins inmonomer, dimer, trimer, tetramer, pentamer, hexamer, or other multimericforms. CTLA4-Ig molecules can comprise a protein fusion with at least anextracellular domain of CTLA4 and an immunoglobulin constant region.CTLA4-Ig molecules can have wild-type or mutant sequences, for example,with respect to the CTLA4 extracellular domain and immunoglobulinconstant region sequences. CTLA4-Ig monomers, alone, or in dimer,tetramer or other multimer form, can be glycosytated.

In some embodiments, the invention provides populations of CTLA4-Igmolecules that have at least a certain percentage of dimer or othermultimer molecules. For example, the invention provides CTLA4-Igmolecule populations that are greater than 90%, 95%, 96%, 97%, 98%, 99%,or 99.5% CTLA4-Ig dimers. In one embodiment, the invention provides aCTLA4-Ig molecule population that comprises from about 95% to about99.5% CTLA4-Ig dimer and from about 0.5% to about 5% of CTLA4-Igtetramer. In another embodiment, the CTLA4-Ig molecule populationcomprises about 98% CTLA4-Ig dimer, about 1.5% CTLA4-Ig tetramer andabout 0.5% CTLA4-Ig monomer.

In one embodiment, the invention provides a population of CTLA4-Igmolecules wherein the population is substantially free of CTLA4-Igmonomer molecules. Substantially free of CTLA4-Ig monomer molecules canrefer to a population of CTLA4-Ig molecules that have less than 1%,0.5%, or 0.1% of monomers.

In one embodiment, the invention provides a population of CTLA4-Igmolecules wherein the population is substantially free of CTLA4-Igmultimers that are larger than dimers, such as tetramers, hexamers, etc.Substantially free of CTLA4-Ig multimer molecules larger than dimers canrefer to a population of CTLA4-Ig molecules that have less than 6%, 5%,4%, 3%, 2%, 1%, 0.5%, or 0.1% of CTLA4-Ig multimers larger than dimericform.

In one embodiment, a CTLA4-Ig monomer molecule can have, for example,the amino acid sequence of: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 ofSEQ ID NO:2 (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ ID NO:2,or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2.When an expression cassette comprising the nucleic acid sequence of SEQID NO: 1 is expressed in CHO cells, the predominant monomer formexpressed has the N-terminus amino acid residue of methionine (residue27 of SEQ ID NO:2), which corresponds to the N-terminus amino acidresidue of wild-type human CTLA4. However, because SEQ ID NO:1 alsoincludes the coding sequence for an Oncostatin M Signal Sequence(nucleotides 11-88 of SEQ ID NO: 1), the expressed protein from SEQ IDNO:1 contains an Oncostatin M Signal Sequence. The signal sequence iscleaved from the expressed protein during the process of protein exportfrom the cytoplasm, or secretion out of the cell. But cleavage canresult in N-terminal variants, such as cleavage between amino acidresidues 25 and 26 (resulting in an N-terminus of residue 26, i.e., the“Ala variant”), or between amino acid residues 24 and 25 (resulting inan N-terminus of residue 2, i.e., the “Met-Ala variant”), as opposed tocleavage between amino acid residues 26 and 27 (resulting in anN-terminus of residue 27). For example, the Met-Ala variant can bepresent in a mixture of CTLA4-Ig molecules at about 1%, and the Alavariant can be present in a mixture of CTLA4-Ig molecules at about8-10%. In addition, the expressed protein from SEQ ID NO:1 can haveC-terminus variants due to incomplete processing. The predominantC-terminus is the glycine at residue 382 of SEQ ID NO:2. In a mixture ofCTLA4-Ig molecules, monomers having lysine at the C-terminus (residue383 of SEQ ID NO:2) can be present, for example, at about 4-5%.

A CTLA4-Ig monomer molecule can comprise an extracellular domain ofhuman CTLA4. In one embodiment, the extracellular domain can comprisethe nucleotide sequence of nucleotides 89-463 of SEQ ID NO:1 that codefor amino acids 27-151 of SEQ ID NO:2. In another embodiment, theextracellular domain can comprise mutant sequences of human CTLA4. Inanother embodiment, the extracellular domain can comprise nucleotidechanges to nucleotides 89-463 of SEQ ID NO:1 such that conservativeamino acid changes are made. In another embodiment, the extracellulardomain can comprise a nucleotide sequence that is at least 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 89-463 ofSEQ ID NO:1.

A CTLA4-Ig monomer molecule can comprise a constant region of a humanimmunoglobulin. This constant region can be a portion of a constantregion; this constant region can have a wild-type or mutant sequence.The constant region can be from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1,IgA2, IgD or IgE. The constant region can be from a light chain or aheavy chain of an immunoglobulin. Where the constant region is from anIgG, IgD, or IgA molecule, the constant region can comprise one or moreof the following constant region domains: CL, CH1, hinge, CH2, or CH3.Where the constant region is from IgM or IgE, the constant region cancomprise one or more of the following constant region domains: CL, CH1,CH2, CH3, or Ca4. In one embodiment, the constant region can comprise onor more constant region domains from IgG, IgD, IgA, IgM or IgE.

In one embodiment, a CTLA4-Ig monomer molecule comprises a modifiedhuman IgG1 hinge region (nucleotides 464-508 of SEQ ID NO: 1; aminoacids 152-166 of SEQ ID NO:2) wherein the serines at amino acid residues156, 162, and 165 of SEQ ID NO:2 have been engineered from cysteinespresent in the wild-type sequence.

In one embodiment, a CTLA4-Ig monomer molecule comprises a modifiedhuman IgG1 CH2 region and a wild-type CH3 region (the modified humanIgG1 CH2 domain having nucleotides 509-838 of SEQ ID NO: 1 and aminoacids 167-276 of SEQ ID NO:2; the human IgG1 CH3 domain havingnucleotides 839-1159 of SEQ ID NO:1 and amino acids 277-383 of SEQ IDNO:2).

In one embodiment, a CTLA4-Ig molecule population comprises monomershaving a sequence shown in any one or more of FIG. 7, 8, or 9 of theU.S. Pat. No. 7,094,874, issued on Aug. 22, 2006 and in U.S. patentapplications published as Publication No. US20030083246 andUS20040022787, which are hereby incorporated by reference in itsentirety.

In one embodiment, a CTLA4-Ig tetramer molecule comprises two pairs ortwo dimers of CTLA4-Ig polypeptides, wherein each polypeptide has one ofthe following amino acid sequences: (i) 26-383 of SEQ ID NO:2, (ii)26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 ofSEQ ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 ofSEQ ID NO:2. Each member of the pair of polypeptides or dimer iscovalently linked to the other member, and the two pairs of polypeptidesare non-covalently associated with one another thereby forming atetramer. Such tetramer molecules are capable of binding to CD80 orCD86.

In another embodiment, such tetramer molecules can bind to CD80 or CD86with an avidity that is at least 2-fold greater than the binding avidityof a CTLA4-Ig dimer (whose monomers have one of the above amino acidsequences) to CD80 or CD86. In another embodiment, such tetramermolecules can bind to CD80 or CD86 with an avidity that is at least2-fold greater than the binding affinity or avidity of wild-type CTLA4to CD80 or CD86. Such greater avidity can contribute to higher efficacyin treating immune disorders and other diseases as described below. Inaddition, greater or improved avidity can produce the result of higherpotency of a drug. For example, a therapeutic composition comprisingCTLA4-Ig tetramer would have a higher avidity and therefore higherpotency than the same amount of a therapeutic composition havingCTLA4-Ig monomer. In another embodiment, such tetramer molecules canhave at least a 2-fold greater inhibition on T cell proliferation ascompared to a CTLA4-Ig dimer (whose monomers have one of the above aminoacid sequences). In another embodiment, such tetramer molecules can haveat least a 2-fold greater inhibition on T cell proliferation as comparedto a wild-type CTLA4 molecule.

T cell proliferation can be measured using standard assays known in theart. For example, one of the most common ways to assess T cellproliferation is to stimulate T cells via antigen or agonisticantibodies to TCR and to measure, for example, the incorporation oftitrated thymidine (3H-TdR) in proliferating T cells or the amount ofcytokines released by proliferating T cells into culture. The inhibitoryeffect of CTLA4-Ig molecules upon T cell activation or proliferation canthereby be measured.

The affinity of a CTLA4-Ig molecule is the strength of binding of themolecule to a single ligand, including CD80, CD86, or CD8OIg or CD86Igfusion proteins. The affinity of CTLA4-Ig to ligands can be measured byusing, for example, binding interaction analysis (BIA) based on surfaceplasmon technique. Aside from measuring binding strength, it permitsreal time determination of binding kinetics, such as association anddissociation rate constants. A sensor chip, consisting of a glass slidecoated with a thin metal film, to which a surface matrix is covalentlyattached, is coated with one of the interactants, i.e, CTLA4-Ig or oneof the ligands. A solution containing the other interactant is allowedto flow over its surface. A continuous light beam is directed againstthe other side of the surface, and its reflection angle is measured. Onbinding of CTLA4-Ig to the ligand, the resonance angle of the light beamchanges (as it depends on the refractive index of the medium close tothe reactive side of the sensor, which in turn is directly correlated tothe concentration of dissolved material in the medium). It issubsequently analyzed with the aid of a computer.

In one embodiment, CTLA4-Ig binding experiments can be performed bysurface plasmon resonance (SPR) on a BIAcore instrument (BIAcore AG,Uppsala, Sweden). CTLA4-Ig can be covalently coupled by primary aminegroups to a carboxymethylated dextran matrix on a BIAcore sensor chip,thereby immobilizing CTLA4-Ig to the sensor chip. Alternatively, ananti-constant region antibody can be used to immobilize CTLA4-Igindirectly to the sensor surface via the Ig fragment. Thereafter,ligands are added to the chip to measure CTLA4-Ig binding to theligands. Affinity measurements can be performed, for example, asdescribed in van der Merwe, P. et al., J. Exp. Med. (1997) 185(3):393-404.

The avidity of CTLA4-Ig molecules can also be measured. Avidity can bedefines as the sum total of the strength of binding of two molecules orcells to one another at multiple sites. Avidity is distinct fromaffininty which is the strength of binding one site on a molecule to itsligand. Without being bound by theory, higher avidity of CTLA4-Igmolecules can lead to increased potency of inhibiton by CTLA4-Igmolecules on T-cell proliferation and activation. Avidity can bemeasured, for example, by two categories of solid phase assays: a)competitive inhibition assays, and b) elution assays. In both of themthe ligand is attached to a solid support. In the competitive inhibitionassay, CTLA4-Ig molecules are then added in solution at a fixedconcentration, together with free ligand in different concentrations,and the amount of ligand which inhibits solid phase binding by 50% isdetermined. The less ligand needed, the stronger the avidity. In elutionassays, the ligand is added in solution. After obtaining a state ofequilibrium, a chaotrope or denaturant agent (e.g. isothiocyanate, urea,or diethylamine) is added in different concentrations to disruptCTLA4-Ig/ligand interactions. The amount of CTLA4-Ig resisting elutionis determined thereafter with an ELISA. The higher the avidity, the morechaotropic agent is needed to elute a certain amount of CTLA4-Ig. Therelative avidity of a heterogeneous mixture of CTLA4-Ig molecules can beexpressed as the avidity index (AI), equal to the concentration ofeluting agent needed to elute 50% of the bound CTLA4-Ig molecules.Refined analysis of data can be performed by determining percentages ofeluted CTLA4-Ig at different concentrations of the eluting agent.

Methods for Producing the CTLA4Ig Molecules of the Invention

Expression of CTLA4Ig molecules can be in prokaryotic cells. Prokaryotesmost frequently are represented by various strains of bacteria. Thebacteria may be a gram positive or a gram negative. Typically,gram-negative bacteria such as E. coli are preferred. Other microbialstrains may also be used.

Sequences, described above, encoding CTLA4Ig molecules can be insertedinto a vector designed for expressing foreign sequences in prokaryoticcells such as E. coli. These vectors can include commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, include such commonly usedpromoters as the beta-lactamase (penicillinase) and lactose (lac)promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan(trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res.8:4057) and the lambda derived P_(L) promoter and N-gene ribosomebinding site (Shimatake, et al., (1981) Nature 292:128).

Such expression vectors will also include origins of replication andselectable markers, such as a beta-lactamase or neomycinphosphotransferase gene conferring resistance to antibiotics, so thatthe vectors can replicate in bacteria and cells carrying the plasmidscan be selected for when grown in the presence of antibiotics, such asampicillin or kanamycin.

The expression plasmid can be introduced into prokaryotic cells via avariety of standard methods, including but not limited to CaCl₂-shock(Cohen, (1972) Proc. Natl. Acad. Sci. USA 69:2110, and Sambrook et al.(eds.), “Molecular Cloning: A Laboratory Manual”, 2nd Edition, ColdSpring Harbor Press, (1989)) and electroporation.

In accordance with the practice of the invention, eukaryotic cells arealso suitable host cells. Examples of eukaryotic cells include anyanimal cell, whether primary or immortalized, yeast (e.g., Saccharomycescerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), and plantcells. Myeloma, COS and CHO cells are examples of animal cells that maybe used as hosts. Particular CHO cells include, but are not limited to,DG44 (Chasin, et la., 1986 Som. Cell. Molec. Genet. 12:555-556; Kolkekar1997 Biochemistry 36:10901-10909), CHO-K1 (ATCC No. CCL-61), CHO-K1Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR,Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B(GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR,Salisbury, Wiltshire, UK), and RR-CHOK1 designated ECACC 92052129 (CAMR,Salisbury, Wiltshire, UK). Illustrative plant cells include tobacco(whole plants, cell culture, or callus), corn, soybean, and rice cells.Corn, soybean, and rice seeds are also acceptable.

Nucleic acid sequences encoding CTLA4Ig molecules described above canalso be inserted into a vector designed for expressing foreign sequencesin a eukaryotic host. The regulatory elements of the vector can varyaccording to the particular eukaryotic host.

Commonly used eukaryotic control sequences for use in expression vectorsinclude promoters and control sequences compatible with mammalian cellssuch as, for example, CMV promoter (CDM8 vector) and avian sarcoma virus(ASV) (πLN vector). Other commonly used promoters include the early andlate promoters from Simian Virus 40 (SV40) (Fiers, et al., (1973) Nature273:113), or other viral promoters such as those derived from polyoma,Adenovirus 2, and bovine papilloma virus. An inducible promoter, such ashMTII (Karin, et al., (1982) Nature 299:797-802) may also be used.

Vectors for expressing CTLA4Ig molecules in eukaryotes may also carrysequences called enhancer regions. These are important in optimizinggene expression and are found either upstream or downstream of thepromoter region.

Examples of expression vectors for eukaryotic host cells include, butare not limited to, vectors for mammalian host cells (e.g., BPV-1, pHyg,pRSV, pSV2, pTK2 (Maniatis); pIRES (Clontech); pRc/CMV2, pRc/RSV, pSFV1(Life Technologies); pVPakc Vectors, pCMV vectors, pSGS vectors(Stratagene)), retroviral vectors (e.g., pFB vectors (Stratagene)),pCDNA-3 (Invitrogen) or modified forms thereof, adenoviral vectors;Adeno-associated virus vectors, baculovirus vectors, yeast vectors(e.g., pESC vectors (Stratagene)).

Nucleic acid sequences encoding CTLA4Ig molecules can integrate into thegenome of the eukaryotic host cell and replicate as the host genomereplicates. Alternatively, the vector carrying CTLA4Ig molecules cancontain origins of replication allowing for extrachromosomalreplication.

For expressing the nucleic acid sequences in Saccharomyces cerevisiae,the origin of replication from the endogenous yeast plasmid, the 2μcircle can be used. (Broach, (1983) Meth. Enz. 101:307). Alternatively,sequences from the yeast genome capable of promoting autonomousreplication can be used (see, for example, Stinchcomb et al., (1979)Nature 282:39); Tschemper et al., (1980) Gene 10:157; and Clarke et al.,(1983) Meth. Enz. 101:300).

Transcriptional control sequences for yeast vectors include promotersfor the synthesis of glycolytic enzymes (Hess et al., (1968) J. Adv.Enzyme Reg. 7:149; Holland et al., (1978) Biochemistry 17:4900).Additional promoters known in the art include the CMV promoter providedin the CDM8 vector (Toyama and Okayama, (1990) FEBS 268:217-221); thepromoter for 3-phosphoglycerate kinase (Hitzeman et al., (1980) J. Biol.Chem. 255:2073), and those for other glycolytic enzymes.

Other promoters are inducible because they can be regulated byenvironmental stimuli or the growth medium of the cells. These induciblepromoters include those from the genes for heat shock proteins, alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, enzymes associatedwith nitrogen catabolism, and enzymes responsible for maltose andgalactose utilization.

Regulatory sequences may also be placed at the 3′ end of the codingsequences. These sequences may act to stabilize messenger RNA. Suchterminators are found in the 3′ untranslated region following the codingsequences in several yeast-derived and mammalian genes.

Illustrative vectors for plants and plant cells include, but are notlimited to, Agrobacterium T_(i) plasmids, cauliflower mosaic virus(CaMV), and tomato golden mosaic virus (TGMV).

Mammalian cells can be transformed by methods including but not limitedto, transfection in the presence of calcium phosphate, microinjection,electroporation, or via transduction with viral vectors.

Methods for introducing foreign DNA sequences into plant and yeastgenomes include (1) mechanical methods, such as microinjection of DNAinto single cells or protoplasts, vortexing cells with glass beads inthe presence of DNA, or shooting DNA-coated tungsten or gold spheresinto cells or protoplasts; (2) introducing DNA by making cell membranespermeable to macromolecules through polyethylene glycol treatment orsubjection to high voltage electrical pulses (electroporation); or (3)the use of liposomes (containing cDNA) which fuse to cell membranes.

US patent application US Publication Number 20050019859 and US patentapplication US Publication Number 20050084933 teach processes for theproduction of proteins of the invention, specifically recombinantglycoprotein products, by animal or mammalian cell cultures and areherein incorporated by reference.

Following the protein production phase of the cell culture process,CTLA4Ig molecules are recovered from the cell culture medium usingtechniques understood by one skilled in the art. In particular, theCTLA4Ig molecule is recovered from the culture medium as a secretedpolypeptide.

The culture medium is initially centrifuged to remove cellular debrisand particulates. The desired protein subsequently is purified fromcontaminant DNA, soluble proteins, and polypeptides, with the followingnon-limiting purification procedures well-established in the art:SDS-PAGE; ammonium sulfate precipitation; ethanol precipitation;fractionation on immunoaffinity or ion-exchange columns; reverse phaseHPLC; chromatography on silica or on an anion-exchange resin such as QAEor DEAE; chromatofocusing; gel filtration using, for example, SephadexG-75™ column; and protein A Sepharose™ columns to remove contaminantssuch as IgG. Addition of a protease inhibitor, such as phenyl methylsulfonyl fluoride (PMSF), or a protease inhibitor cocktail mix also canbe useful to inhibit proteolytic degradation during purification. Aperson skilled in the art will recognize that purification methodssuitable for a protein of interest, for example a glycoprotein, canrequire alterations to account for changes in the character of theprotein upon expression in recombinant cell culture.

Purification techniques and methods that select for the carbohydrategroups of the glycoprotein are also of utility within the context of thepresent invention. For example, such techniques include, HPLC orion-exchange chromatography using cation- or anion-exchange resins,wherein the more basic or more acidic fraction is collected, dependingon which carbohydrate is being selected for. Use of such techniques alsocan result in the concomitant removal of contaminants.

The purification method can further comprise additional steps thatinactivate and/or remove viruses and/or retroviruses that mightpotentially be present in the cell culture medium of mammalian celllines. A significant number of viral clearance steps are available,including but not limited to, treating with chaotropes such as urea orguanidine, detergents, additional ultrafiltration/diafiltration steps,conventional separation, such as ion-exchange or size exclusionchromatography, pH extremes, heat, proteases, organic solvents or anycombination thereof.

The purified CTLA4Ig molecule require concentration and a bufferexchange prior to storage or further processing. A Pall Filtron TFFsystem may be used to concentrate and exchange the elution buffer fromthe previous purification column with the final buffer desired for thedrug substance.

In one aspect, purified CTLA4Ig molecules, which have been concentratedand subjected to diafiltration step, can be filled into 2-L Biotainer®bottles, 50-L bioprocess bag or any other suitable vessel. CTLA4Igmolecules in such vessels can be stored for about 60 days at 2° to 8° C.prior to freezing. Extended storage of purified CTLA4Ig molecules at 2°to 8° C. may lead to an increase in the proportion of HMW species.Therefore, for long-term storage, CTLA4Ig molecules can be frozen atabout −70° C. prior to storage and stored at a temperate of about −40°C. The freezing temperature can vary from about −50° C. to about −90° C.The freezing time can vary and largely depends on the volume of thevessel that contains CTLA4Ig molecules, and the number of vessels thatare loaded in the freezer. For example, in one embodiment, CTLA4Igmolecules are in 2-L Biotainer® bottles. Loading of less than four 2-LBiotainer® bottles in the freezer may require from about 14 to at least18 hours of freezing time. Loading of at least four bottles may requirefrom about 18 to at least 24 hours of freezing time. Vessels with frozenCTLA4Ig molecules are stored at a temperature from about −35° C. toabout −55° C. The storage time at a temperature of about −35° C. toabout −55° C. can vary and can be as short as 18 hours. The frozen drugsubstance can be thawed in a control manner for formulation of drugproduct.

Co-pending U.S. patent application Ser. No. 60/752,267, filled on Dec.20, 2005 and Ser. No. 06/849,543, filed on Oct. 5, 2006 and PCT patentapplication Attorney Docket No. 10734 PCT titled Cell Lines,Compositions and Methods for Producing a Composition with the inventorKirk Leister filed on Dec. 19, 2006 teach processes for the productionof proteins of the invention, specifically recombinant glycoproteinproducts, by animal or mammalian cell cultures and is hereinincorporated by reference.

Lyophilized Formulation of the Invention

The lyophilized formulation of the invention comprises the CTLA4Igmolecule in a weight ratio of at least 1:2 protein to lyoprotectant. Thelyoprotectant is preferably sugar, more preferably disaccharides, mostpreferably sucrose or maltose. The lyophilized formulation may alsocomprise one or more of the components selected from the list consistingof buffering agents, surfactants, bulking agents and preservatives.

During formulation development studies, the effects of variousexcipients on the solution- and freeze-dried solid-state stability ofthe CTLA4Ig molecule were evaluated.

Instability of the freeze-dried CTLA4Ig molecule in the absence of astabilizer clearly highlighted a need for inclusion of a lyoprotectantin the formulation. Initial screening studies showed that drug productwas stable in the presence of sugars such as maltose and sucrose andamino acids such as arginine and lysine. Polyols such as mannitol andpolysaccharides such as dextran 40 were detrimental to its stability.The preferred lyoprotectants are the disaccharides maltose and sucrose.

Example VI describes stability studies of freeze dried Abatacept drugproduct in the presence of maltose stored at 50° C. An increase in highmolecular weight (HMW) species was monitored using astability-indicating size exclusion chromatography (SE-HPLC) assay. Theresults demonstrate that the stability of the CTLA4Ig molecule in afreeze-dried solid-state form is enhanced in the presence of maltose.

The amount of sucrose or maltose useful for stabilization of thelyophilized drug product is in a weight ratio of at least 1:2 protein tosucrose or maltose, preferably in a weight ratio of from 1:2 to 1:5protein to sucrose or maltose, more preferably in a weight ratio ofabout 1:2 protein to maltose or sucrose.

If necessary, the pH of the formulation, prior to lyophilization, is setby addition of a pharmaceutically acceptable acid and/or base. Thepreferred pharmaceutically acceptable acid is hydrochloric acid. Thepreferred base is sodium hydroxide.

During formulation development, the stability of the freeze-dried drugproduct was studied as a function of pH. Example VI describes stabilitystudies with freeze dried Abatacept drug product as a function of pH.The solution pH was adjusted between 6 to 8 prior to freeze-drying. Thesamples were placed on stability and the constituted product vials weremonitored for an increase in the high molecular weight species atvarious time points using a stability-indicating size-exclusionchromatography (SE-HPLC) assay. Under the recommended storage conditionof 2°-8° C., no significant changes in the rate of formation of HMWspecies were observed. Additionally; the solution-state stability datagenerated during an early development showed the pH of maximum stabilityto be between 7 and 8. The acceptable pH range for the lyophilized drugproduct is from 7 to 8 with a preferred target pH of 7.5.

In another aspect, the salts or buffer components may be added in anamount of at least about 10 mM, preferably 10-200 mM. The salts and/orbuffers are pharmaceutically acceptable and are derived from variousknown acids (inorganic and organic) with “base forming” metals oramines. In addition to phosphate buffers, there can be used glycinate,carbonate, citrate buffers and the like, in which case, sodium,potassium or ammonium ions can serve as counterion.

A “bulking agent” is a compound which adds mass to a lyophilized mixtureand contributes to the physical structure of the lyophilized cake (e.g.facilitates the production of an essentially uniform lyophilized cakewhich maintains an open pore structure). Illustrative bulking agentsinclude mannitol, glycine, polyethylene glycol and sorbitol. Thelyophilized formulations of the present invention may contain suchbulking agents

A preservative may be optionally added to the formulations herein toreduce bacterial action. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation.

One skilled in the art would select the amount of drug product to befiled into a vial depending on the required dosages and administrationschedule for a specific treatment. For example, the concentration ofCTLA4Ig per vial may range from 50 to 300 mg/vial, preferably 100 to 250mg/vial.

A typical composition of lyophilized Abatacept drug product is listed inTable 1 below.

TABLE 1 Composition of lyophilized abatacept (250 mg/vial) drug productComponent Amount (mg/vial)^(a) Abatacept 262.5 Maltose monohydrate 525Sodium phosphate monobasic, monohydrate^(b) 18.1 Sodium chloride^(b)15.3 Hydrochloric Acid Adjust to pH 7.5 Sodium hydroxide Adjust to pH7.5 ^(a)includes a 5% overfill for vial, needle, syringe loss ^(b)Thesecomponents are present in the abatacept drug substance solution

A typical composition of lyophilized Belatacept drug product is listedin Table 2 below.

TABLE 2 Composition of lyophilized Belatacept 100 mg/vial drug productComponent Amount/Vial (mg)^(a) Belatacept 110^(a)   Sucrose 220   Sodium Phosphate Monobasic Monohydrate 15.18 Sodium Chloride  2.55 1NSodium Hydroxide Adjust to pH 7.5 1N Hydrochloric Acid Adjust to pH 7.5^(a)Each vial contains 10% overfill for vial, needle and syringe holdupof the reconstituted solution.

The lyophilized drug product is constituted with an aqueous carrier. Theaqueous carrier of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, afterlyophilization. Illustrative diluents include sterile water forinjection (SWFI), bacteriostatic water for injection (BWFI), a pHbuffered solution (e.g. phosphate-buffered saline), sterile salinesolution, Ringer's solution or dextrose solution.

Preferably, the lyophilized drug product of the current invention isconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9%Sodium Chloride Injection, USP. During constitution, the lyophilizedpowder rapidly dissolves providing a clear, colorless solution.

Typically, the lyophilized drug product of the instant invention isconstituted to about 25 mg/ml with 10 ml of either Sterile Water forInjection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. Theconstituted solution is further diluted to drug product concentrationsbetween 1 and 10 mg/ml with 0.9% Sodium Chloride Injection, USP. Thediluted drug product for injection is isotonic and suitable foradministration by intravenous infusion.

During early clinical development, the constituted solutions oflyophilized drug product were found to be incompatible with disposablesiliconized syringes, which are commonly used for the preparation andadministration of the parenteral products. Specifically, the constituteddrug product solution developed thread-like, gelatinous particles whenstored in these syringes for more than 10-15 minutes. Further studiesdemonstrated that this incompatibility was due to the interaction ofdrug product with silicone oil (dimethylsiloxane), which is used as alubricant on these syringes. The formation of these particles did notaffect the potency of the drug product solution over its use time.Silicone free syringes are preferably utilized for drug productconstitution and transfer of the constituted solutions from the vial tothe intravenous bag.

Alternatively, a surfactant may be added to the formulation to reduce orprevent the interaction of the constituted drug product with thesiliconized syringe, for example, in an amount sufficient thereof.

The recommended storage condition for the lyophilized formulation isfrom 2-8° C. with a recommended shelf life of at least 12 months.

Preparation of the Lyophilized Formulation

The lyophilized drug product manufacturing process involves batching ofthe Formulated Bulk Solution for lyophilization, aseptic filtration,filling in vials, freezing vials in a freeze-dryer chamber, followed bylyophilization, stoppering and capping.

Examples III and IV describe the manufacture of the lyophilizedAbatacept and Belatacept drug products, respectively.

The Formulated Bulk Solution is typically set at a fixed proteinconcentration so that the desired vial fill volume can be kept constant.During the addition of lyoprotectant, the mixer speed is controlled at250±50 rpm so as to minimize foaming in the batching solution and toensure complete dissolution of the lyoprotectant within 10 to 20minutes. The Formulated Bulk Solution can be stored under 2°-8° C. orroom temperature and room light for at least 32 hours prior tolyophilization.

The Formulated Bulk Solution is not terminally sterilized due to heatsensitivity of the CTLA4Ig molecule. The Formulated Bulk Solution may besterilized using two 0.22-μm Millipore Millipak® sterilizing gradefilters in series prior to filling in vials for lyophilization.

The freeze-drying cycle selected for this product is optimized in orderto have efficient sublimation and evaporation during the primary andsecondary phases of drying respectively, without compromising theproduct quality.

To aid in rapid dissolution of the lyophilized powder and to prevent theformation of excessive foam during constitution of the drug product, thelyophilized vials are stoppered under 500±100 microns chamber pressureat the end of the freeze-drying cycle.

One skilled in the art would be aware of the need to overfill thecontainer so as to compensate for vial, needle, syringe hold-up duringpreparation and injection. For example, each vial of Abatacept drugproduct, 250 mg/ml, contains a 5% overage of drug product to account forreconstitution and withdrawal losses.

Liquid Subcutaneous Formulation

One skilled in the art would recognize the inconvenience of an IVformulation for the patient in need of frequent, chronic therapy. Thepatient has to make frequent trips to the hospital to receive their drugvia an IV infusion that may last as long as an hour. Consequently, a SCformulation that could be self-administered at home would be verybeneficial to such a patient.

For subcutaneous administration, a dosage form with high proteinconcentrations is desired. Treatments with high doses of more than 1mg/kg (>100 mg per dose) require development of formulations atconcentrations exceeding 100 mg/ml because of the small volume (<1.5 ml)that can be given by the SC routes. In order to optimize the long termstability at high concentration against formation of high molecularweight species, formulation development studies were conducted toevaluate the effect of various excipients on the solution statestability of the liquid SC formulations of the invention.

The SC formulation of the invention comprises the CTLA4Ig molecule at aprotein concentration of at least 100 mg/ml in combination with a sugarat stabilizing levels, preferably a protein concentration of at least125 mg/ml in combination with a sugar at stabilizing levels, in anaqueous carrier. The sugar is preferably in a weight ratio of at least1:1.1 protein to sugar. The stabilizer is preferably employed in anamount no greater than that which may result in a viscosity undesirableor unsuitable for administration via SC syringe. The sugar is preferablydisaccharides, most preferably sucrose. The SC formulation may alsocomprise one or more of the components selected from the list consistingof buffering agents, surfactants, and preservatives.

The stability profile of the SC formulation was evaluated in presence ofvarious stabilizers including sugars, polysaccharides, amino acids,surfactants, polymers, cyclodextrans, proteins, etc. Among all theexcipients evaluated, sugars such as sucrose, mannitol and trehalose hada better stabilizing effect against the formation of high molecularweight species.

Examples V and VIII describe stability studies of SC Belatacept andAbatacept drug product, respectively, in the presence of sucrose storedat various temperatures for various time periods. An increase in highmolecular weight species was monitored using a stability-indicating sizeexclusion chromatography (SE-HPLC) assay. The results demonstrate thatthe stability of the CTLA4Ig molecule in the SC formulation is enhancedin the presence of sucrose. Stabilization by sucrose was better athigher sucrose:protein weight ratio. Based on these studies, sucrose wasselected as the stabilizer at a ratio that provides optimum stabilitywithout resulting in a SC solution with excessive hypertonicity.

The amount of sucrose useful for stabilization of the SC drug product isin a weight ratio of at least 1:1 protein to sucrose, preferably in aweight ratio of from 1:1.3 to 1:5 protein to sucrose, more preferably ina weight ratio of about 1:1.4 protein to sucrose.

If necessary, the pH of the formulation is set by addition of apharmaceutically acceptable acid and/or base. The preferredpharmaceutically acceptable acid is hydrochloric acid. The preferredbase is sodium hydroxide.

During formulation development, the stability of the SC drug product wasstudied as a function of pH. Example V describes stability studies withSC Belatacept drug product as a function of pH. The SC formulation pHwas adjusted between 7 to 8.2, samples were placed on stability and thedrug product was monitored for an increase in the high molecular weightspecies at various time points using a stability-indicatingsize-exclusion chromatography (SE-HPLC) assay. Under the recommendedstorage condition of 2°-8° C., no significant changes in the rate offormation of HMW species were observed after 3 months.

In addition to aggregation, deamidation is a common product variant ofpeptides and proteins that may occur during fermentation, harvest/cellclarification, purification, drug substance/drug product storage andduring sample analysis. Deamidation is the loss of NH₃ from a proteinforming a succinimide intermediate that can undergo hydrolysis. Thesuccinimide intermediate results in a 17 u mass decrease of the parentpeptide. The subsequent hydrolysis results in an 18 u mass increase.Isolation of the succinimide intermediate is difficult due toinstability under aqueous conditions. As such, deamidation is typicallydetectable as 1 u mass increase. Deamidation of an asparagine results ineither aspartic or isoaspartic acid. The parameters affecting the rateof deamidation include pH, temperature, solvent dielectric constant,ionic strength, primary sequence, local polypeptide conformation andtertiary structure. The amino acid residues adjacent to Asn in thepeptide chain affect deamidation rates. Gly and Ser following an Asn inprotein sequences results in a higher susceptibility to deamidation.

Preliminary laboratory scale stability studies suggest that deamidationwill exceed reference levels of the peptide mapping test method at 24months using the SC abatacept formulation at pH 7.8. The data at sixmonths under both 2-8° C. and 25° C. at 60% humidity showed that therate of demaidation was lower at pH 7.2 and higher at pH 8 when comparedto the SC abatacept sample at pH 7.8. Examples IX and XII describelaboratory scale pH studies designed to evaluate deamidation in SC drugproduct formulations in the pH range of 6.3 to 7.2.

The acceptable pH range for the SC drug product is from 6 to 8,preferably 6 to 7.8, more preferably 6 to 7.2.

In another aspect, the salts or buffer components may be added in anamount of at least 10 mM, preferably 10-200 mM. The salts and/or buffersare pharmaceutically acceptable and are derived from various known acids(inorganic and organic) with “base forming” metals or amines. Inaddition to phosphate buffers, there can be used glycinate, carbonate,citrate buffers and the like, in which case, sodium, potassium orammonium ions can serve as counterion.

Example VIII describes the effect of buffer strength on Abatacept SCdrug product. Stability was better in 10 mM phosphate buffer compared to5 mM phosphate buffer at pH 7.5 at 100 mg/mL abatacept drug productconcentration. Moreover the higher buffering capacity of 10 mM phosphatebuffer offered better pH control of the formulation compared to 5 mMbuffer.

The aqueous carrier of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation. Illustrativecarriers include sterile water for injection (SWFI), bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

A preservative may be optionally added to the formulations herein toreduce bacterial action. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation.

As discussed with the lyophilized drug product, the CTLA4Ig molecule isincompatible with silicone found in standard syringes, in that, itinteracts with silicone to form visible particulates, thereby limitingthe patient to utilizing silicone free syringes. The SC formulation mayoptionally comprise a surfactant to prevent formation of visibleparticulates in presence of silicone

Examples V and VIII describe the effect of surfactants such asPolysorbate 80 and Poloxamer 188 on the solution stability of belataceptand abatacept drug product, respectively, and it was found thatsurfactants did not have an effect on the stability of the CTLA4Igmolecule in a SC formulation. Different levels of Poloxamer 188 wereevaluated and the concentration of from 4 mg/ml to 8 mg/ml, preferable 8mg/ml was found to be adequate to prevent the silicone relatedparticulate formation in the formulation.

A typical composition of belatacept SC drug product, 125 mg/ml (100mg/vial) drug product is provided in Table 3 below.

TABLE 3 Composition of Belatacept SC drug product, 125 mg/ml (100mg/vial) Component Amount (mg/vial)^(b) Belatacept 140.0 Sucrose 190.4Poloxamer 188 8.96 Sodium phosphate monobasic, monohydrate 0.371 Sodiumphosphate dibasic, anhydrous 1.193 Water for Injection q.s. to 1.12 ml^(d) Includes 40% overfill for Vial, Needle, Syringe loss

A typical composition of abatacept SC drug product, 125 mg/ml (125mg/vial) is provided in Table 4 below.

TABLE 4 Composition of Abatacept SC drug product, 125 mg/ml (125mg/vial) Component Amount (mg/vial)^(c) Abatacept 175 Sucrose 238Poloxamer 188 11.2 Sodium phosphate monobasic, monohydrate 0.20 Sodiumphosphate dibasic, anhydrous 136 Water for Injection q.s. to 1.4 ml ^(d)Includes 40% overfill for Vial, Needle, Syringe loss

A typical composition of abatacept SC drug product, 125 mg/ml filed intoa syringe is provided in Table 5 below.

TABLE 5 Composition of Abatacept SC drug product, 125 mg/ml (125mg/syringe) Component Amount (mg/syringe) Abatacept 125 Sucrose 170Poloxamer 188 8.0 Sodium phosphate monobasic, monohydrate 0.143 Sodiumphosphate dibasic, anhydrous 0.971 Water for Injection q.s. to 1. ml

The recommended storage condition for the SC formulation is from 2-8° C.with a recommended shelf life of at least 12 months.

The density of the abatacept SC drug product and the matching placebowas determined at ambient temperature using the Mettler-Toledodensitometer. Measurements were performed using 5-mL samples intriplicates. The density of the abatacept SC formulation was found to be1.1 g/cc and that of the placebo product was found to be 1.065 g/cc.Typically, the density of a SC CTLA4Ig formulation is about 1.0 g/cc toabout 1.2 g/cc, preferably about 1.0 g/cc to about 1.15 g/cc, morepreferably about 1.095 g/cc to about 1.105 g/cc.

The viscosity of the abatacept SC formulation was determined using theBrookfield rheometer at ambient temperature. A reference standard of 9.3cps was used for the measurements. The viscosity of the SC drug productat 125 mg/mL abatacept concentration was found to be 13±2 cps.Typically, the viscosity of a SC CTLA4Ig formulation at 125 mg/mL isabout 9 to about 20 cps, preferably about 9 to about 15 cps, morepreferably 12 to about 15 cps.

Osmolality of SC abatacept drug product and placebo formulation wasmeasured using a vapor pressure method. The data show that atconcentrations of 125 mg/mL, the osmolality of abatacept SC formulationis 770±25 mOsm/kgH2O Typically, the osmolatlity of a SC CTLA4Igformulation at 125 mg/mL is about 250 to about 800 mOsm/kgH₂O,preferably about 700 to about 800 mOsm/kgH2O, more preferably about 750to about 800 mOsm/kgH₂O.

Preparation of the SC Formulation

The manufacturing process developed for SC formulations typicallyinvolves compounding with sugar and surfactant, followed by asepticsterile filtration and filling into vials or syringes, optionallypreceded by diafiltration (buffer exchange) and concentration of drugsubstance using an ultrafiltration unit.

Examples I and II describe the manufacture of the SC Belatacept andAbatacept, drug products, respectively.

One skilled in the art would be aware of the need to overfill thecontainer so as to compensate for vial, needle, syringe hold-up duringpreparation and injection. For example, a 40% overage of drug product isincorporated into each vial of SC liquid formulation to account forwithdrawal losses and guarantee that 0.8 ml of the solution containing100 mg of belatacept drug product can be withdrawn from the vial.

Liquid Formulation

IV formulations may be the preferred administration route in particularinstances, such as when a patient is in the hospital aftertransplantation receiving all drugs via the IV route. One skilled in theart would recognize the disadvantages and risks of a lyophilizedformulation for both the manufacturer and the health care professional,respectively. The risks and disadvantages to the health careprofessional associated with reconstitution can include contamination,foaming and product loss as well the health care professional's timerequired to prepare the IV formulation. Additionally, the manufacturer'scosts in equipment and employee time can be decreased by removing thelyophilization step of a manufacturing process. All of these reasons aresufficient motivation to design a liquid formulation for IV use.

The preferred liquid formulation to develop would be a formulation thatwould mimic the lyophilized drug product after the first constitution toa protein concentration of about 25 mg/ml. The purchased liquidformulation would then be further diluted to the desired drug productconcentrations between 1 and 10 mg/ml with 0.9% Sodium ChlorideInjection, USP by the health care professional at time of use. Thediluted drug product for injection is isotonic and suitable foradministration by intravenous infusion.

As discussed above, long term stability of liquid formulations is anissue for protein drug products. In order to confirm the long termstability of a solution against formation of high molecular weightspecies, formulation development studies were conducted to evaluate thesolution state stability of the liquid formulation of the invention.

The liquid formulation of the invention comprises the CTLA4Ig moleculeat a protein concentration of at least 20 mg/ml in combination with asugar at stabilizing levels, preferably at least 25 mg/ml in combinationwith a sugar at stabilizing level in an aqueous carrier. Preferably thesugar is in a weight ratio of at least 1:1 protein to sugar. The sugaris preferably disaccharides, most preferably sucrose. The liquidformulation may also comprise one or more of the components selectedfrom the list consisting of buffering agents, surfactants, andpreservatives.

The amount of sucrose useful for stabilization of the liquid drugproduct is in a weight ratio of at least 1:1 protein to sucrose,preferably in a weight ratio of from 1:2 to 1:10 protein to sucrose,more preferably in a weight ratio of about 1:2 protein to sucrose.

If necessary, the pH of the formulation is set by addition of apharmaceutically acceptable acid and/or base. The preferredpharmaceutically acceptable acid is hydrochloric acid. The preferredbase is sodium hydroxide.

During formulation development, the stability of the liquid drug productwas studied at a target pH of 7.5. Example VII describes stabilitystudies with liquid Belatacept drug product, at a pH of 7.5. The liquidformulation pH was adjusted to 7.5, samples were placed on stability andthe drug product was monitored for an increase in the high molecularweight species at various time points using a stability-indicatingsize-exclusion chromatography (SE-HPLC) assay. Under the recommendedstorage condition of 2°-8° C., no significant changes in the rate offormation of HMW species were observed.

In addition to aggregation, deamidation and fragmentation are productvariant of peptides and proteins that may occur during fermentation,harvest/cell clarification, purification, drug substance/drug productstorage and during sample analysis. Preliminary laboratory scalestability studies suggest that deamidation will exceed reference levelsof the peptide mapping test method (rise above the 5% T26a or T26b (%T30) maximum) and that fragmentation will exceed reference levels forthe SDS-PAGE test method (drop below the 96% major band minimum) at 24months using the belatacept (20 mg/ml at pH 7.5) stored at 2°-8° C. Datafrom liquid formulations (see SC data above) show that the rate ofdeamidation found in the formulations of the invention decreases as thepH of the formulations is lowered.

The acceptable pH range for the liquid drug product is from 6 to 8preferably 6 to 7.8, more preferably 6 to 7.2.

In another aspect, the salts or buffer components may be added in anamount of at least about 10 mM, preferably 10-200 mM. The salts and/orbuffers are pharmaceutically acceptable and are derived from variousknown acids (inorganic and organic) with “base forming” metals oramines. In addition to phosphate buffers, there can be used glycinate,carbonate, citrate buffers and the like, in which case, sodium,potassium or ammonium ions can serve as counterion.

The aqueous carrier of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation. Illustrativecarriers include sterile water for injection (SWFI), bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

A preservative may be optionally added to the formulations herein toreduce bacterial action. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation.

As discussed with the lyophilized drug product, the CTLA4Ig molecule isincompatible with silicone found in standard syringes, in that, itinteracts with silicone to form visible particulates, thereby limitingthe health care professional to utilizing silicone free syringes. Theliquid formulation may optionally comprise a surfactant to preventformation of visible particulates in presence of silicone.

A typical composition of Belatacept liquid drug product, 20 mg/ml (250mg/vial) drug product is provided in Table 6 below.

TABLE 6 Composition of Belatacept liquid drug product, 20 mg/ml (250mg/vial) Component Amount^(a) (mg/vial) Belatacept 260 Sucrose 520Sodium phosphate monobasic, monohydrate 18.1 Sodium chloride 15.3Hydrochloric acid Adjust to pH 7.5 Sodium hydroxide Adjust to pH 7.5Water for Injection q.s. to 13 ml ^(a)includes 4% overfill for vial,needle and syringe losses

The recommended storage condition for the liquid formulation is from2-8° C. with a recommended shelf life at least 12 months.

Preparation of the Liquid Formulation

The liquid drug product manufacturing process typically involvescompounding with sugar and optionally surfactant followed by asepticfiltration and filling in vials, stoppering and capping.

The Formulated Bulk Solution is typically set at a fixed proteinconcentration so that the desired vial fill volume can be kept constant.During the addition of sugar to drug substance, the mixer speed iscontrolled at 250±50 rpm so as to minimize foaming in the batchingsolution and to ensure complete dissolution of the sugar within 10 to 20minutes. The Formulated Bulk Solution can be stored under 2°-8° C. orroom temperature and room light for at least 24 hours prior to filling.

The Bulk Solution is not terminally sterilized due to heat sensitivityof the CTLA4Ig molecule. The Bulk Solution may be sterilized using two0.22-μm Millipore Millipak® sterilizing grade filters in series prior tofilling in vials.

One skilled in the art would be aware of the need to overfill thecontainer so as to compensate for vial, needle, syringe hold-up duringpreparation and injection. For example, each vial of Belatacept drugproduct, 20 mg/mL (250 mg/vial), contains a 4% overage of drug productto account for reconstitution and withdrawal losses.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided which contains the drug product and preferably providesinstructions for its use. The article of manufacture comprises acontainer. Suitable containers include, for example, bottles, vials,syringes and test tubes. The container may be formed from a variety ofmaterials such as glass, plastic or metals.

The container holds the lyophilized or liquid formulations. The labelon, or associated with, the container may indicate directions forreconstitution and/or use. For example, the label may indicate that the25 mg/ml belatacept drug product is to be diluted to proteinconcentrations as described above. The label may further indicate thatthe SC formulation is useful or intended for subcutaneousadministration. The container holding the formulation may be a multi-usevial, which allows for repeat administrations (e.g. from 2-6administrations) of, for example, the subcutaneous formulation.Alternatively, the container may be a pre-filled syringe containing, forexample, the subcutaneous formulation.

The article of manufacture may further comprise a second containercomprising, for example, a suitable carrier for the lyophilizedformulation.

The article of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

Silicone free syringes are preferably utilized for surfactant free drugproduct, such as upon reconstitution of lyophilized drug product and/ortransfer of the solutions from the vial to the intravenous bag and maybe co-packaged with the drug product vial.

Methods of Use

The present invention further provides methods for treating immunesystem diseases or tolerance induction comprising administering aneffective amount of the CTLA4Ig molecule formulations of the invention.In particular embodiments, the immune system diseases are mediated byCD28- and/or CTLA4-positive cell interactions with CD80/CD86-positivecells. In a further embodiment, T cell interactions are inhibited.Immune system diseases include, but are not limited to, autoimmunediseases, immunoproliferative diseases, and graft-related disorders.These methods comprise administering to a subject the CTLA4Ig moleculeformulations of the invention to regulate T cell interactions with theCD80- and/or CD86-positive cells. Examples of graft-related diseasesinclude graft versus host disease (GVHD) (e.g., such as may result frombone marrow transplantation, or in the induction of tolerance), immunedisorders associated with graft transplantation rejection, chronicrejection, and tissue or cell allo- or xenografts, including solidorgans, skin, islets, muscles, hepatocytes, neurons. Examples ofimmunoproliferative diseases include, but are not limited to, psoriasis;T cell lymphoma; T cell acute lymphoblastic leukemia; testicularangiocentric T cell lymphoma; benign lymphocytic angiitis; andautoimmune diseases such as lupus (e.g., lupus erythematosus, lupusnephritis), Hashimoto's thyroiditis, primary myxedema, Graves' disease,pernicious anemia, autoimmune atrophic gastritis, Addison's disease,diabetes (e.g. insulin dependent diabetes mellitis, type I diabetesmellitis), good pasture's syndrome, myasthenia gravis, pemphigus,Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiplesclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia,primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis,Sjogren's syndrome, rheumatic diseases (e.g., rheumatoid arthritis),polymyositis, scleroderma, and mixed connective tissue disease.

The present invention further provides a method for inhibiting solidorgan and/or tissue transplant rejections by a subject, the subjectbeing a recepient of transplant tissue. Typically, in tissuetransplants, rejection of the graft is initiated through its recognitionas foreign by T cells, followed by an immune response that destroys thegraft. The CTLA4Ig molecule formulations of this invention, byinhibiting T lymphocyte proliferation and/or cytokine secretion, mayresult in reduced tissue destruction and induction of antigen-specific Tcell unresponsiveness may result in long-term graft acceptance withoutthe need for generalized immunosuppression. Furthermore, the CTLA4Igmolecule formulations of the invention can be administered with otherpharmaceuticals including, but not limited to, corticosteroids,cyclosporine, rapamycin, mycophenolate mofetil, azathioprine,tacrolismus, basiliximab, and/or other biologics.

The present invention also provides methods for inhibiting graft versushost disease in a subject. This method comprises administering to thesubject the formulations of the invention, alone or together, withfurther additional ligands, reactive with IL-2, IL-4, or γ-interferon.For example, a CTLA4Ig molecule SC formulation of this invention may beadministered to a bone marrow transplant recipient to inhibit thealloreactivity of donor T cells. Alternatively, donor T cells within abone marrow graft may be tolerized to a recipient's alloantigens ex vivoprior to transplantation.

Inhibition of T cell responses by CTLA4Ig molecule formulations of theinvention may also be useful for treating autoimmune disorders. Manyautoimmune disorders result from inappropriate activation of T cellsthat are reactive against autoantigens, and which promote the productionof cytokines and autoantibodies that are involved in the pathology ofthe disease. Administration of a CTLA4Ig molecule formulation in asubject suffering from or susceptible to an autoimmune disorder mayprevent the activation of autoreactive T cells and may reduce oreliminate disease symptoms. This method may also comprise administeringto the subject a formulation of the invention, alone or together, withfurther additional ligands, reactive with IL-2, IL-4, or γ-interferon.

The most effective mode of administration and dosage regimen for theformulations of this invention depends upon the severity and course ofthe disease, the patient's health and response to treatment and thejudgment of the treating physician. In accordance with the practice ofthe invention an effective amount for treating a subject may be betweenabout 0.1 and about 10 mg/kg body weight of subject. Also, the effectiveamount may be an amount between about 1 and about 10 mg/kg body weightof subject.

The CTLA4Ig molecule formulations of the invention may be administeredto a subject in an amount and for a time (e.g. length of time and/ormultiple times) sufficient to block endogenous B7 (e.g., CD80 and/orCD86) molecules from binding their respective ligands, in the subject.Blockage of endogenous B7/ligand binding thereby inhibits interactionsbetween B7-positive cells (e.g., CD80- and/or CD86-positive cells) withCD28- and/or CTLA4-positive cells. Dosage of the CTLA4Ig molecule isdependant upon many factors including, but not limited to, the type oftissue affected, the type of disease being treated, the severity of thedisease, a subject's health, and a subject's response to the treatmentwith the agents. Accordingly, dosages of the agents can vary dependingon the subject and the mode of administration, US patent application USPublication Number US 2003/0083246 and US patent application USPublication Number US 2004/0022787 teach dosage and administrationschedules for CTLA4Ig having the amino acid sequence shown in SEQ IDNO:2 for treating rheumatic diseases, such as rheumatoid arthritis. Allare herein incorporated by reference

An effective amount of CTLA4Ig molecule may be administered to a subjectdaily, weekly, monthly and/or yearly, in single or multiple times perhour/day/week/month/year, depending on need. For example, in oneembodiment, an effective amount of the CTLA4Ig molecule may initially beadministered once every two weeks for a month, and then once every monththereafter.

An effective amount of CTLA4Ig molecule is an amount about 0.1 to 100mg/kg weight of a subject. In another embodiment, the effective amountis an amount about 0.1 to 20 mg/kg weight of a subject. In a specificembodiment, the effective amount of CTLA4Ig is about 2 mg/kg weight of asubject. In another specific embodiment, the effective amount of CTLA4Igis about 10 mg/kg weight of a subject. In another specific embodiment,an effective amount of CTLA4Ig is 500 mg for a subject weighing lessthan 60 kg, 750 mg for a subject weighing between 60-100 kg and 1000 mgfor a subject weighing more than 100 kg.

U.S. patent application Ser. No. 60/668,774, filed Apr. 6, 2005 and U.S.patent application Ser. No. 11/399,666, filed Apr. 6, 2006 teach thedosage and administration schedule for LEA29YIg having the amino acidsequence shown in SEQ ID NO:4 for treating immune disorders associatedwith graft transplantation. All are herein incorporated by reference.

Typically, doses of the CTLA4Ig molecule formulation of the inventionare based on body weight, and administration regimens may be dictated bythe target serum trough profiles. Typically, target trough serumconcentration of LEA29YIg molecules of the invention between about 3μg/mL and about 30 μg/mL over the first 3 to 6 months post-transplantwill be sufficient to maintain function of the allograft, preferablybetween about 5 μg/mL and about 20 μg/mL. Typically, target trough serumconcentration of LEA29YIg molecules of the invention during themaintenance phase are between about 0.2 μg/mL and about 3 μg/mL,preferably between about 0.25 μg/mL and about 2.5 μg/mL.

The LEA29YIg molecules of the invention may be administered in an amountbetween about 0.1 to about 20.0 mg/kg weight of the patient, typicallybetween about 1.0 to about 15.0 mg/kg. For example, L104EA29YIg may beadministered at 10 mg/kg weight of the patient during the early phase,high risk period that follows transplantation and decreased to 5 mg/kgweight of the patient for a maintenance dosage.

The administration of the CTLA4Ig molecules of the invention can be viaa 30 minute to one or more hour intravenous infusion. Alternatively,single to multiple subcutaneous injections can deliver the requireddosage. Typically, a 30 minute intravenous infusion is theadministration route utilized during the early phase of treatment whilethe patient is in the hospital and/or making scheduled visits to thehealthcare professional for monitoring. The subcutaneous injection isthe typical administration mode utilized during the maintenance phase,thereby allowing the patient to return to their normal schedule bydecreasing the visits to a healthcare professional for intravenousinfusions.

Example 10 describes the pharmacokinetics of the lyophilized IV CTLA4Igformulation in healthy subjects and patients with rheumatoid arthritis(RA). The pharmacokinetics of abatacept in RA patients and healthysubjects appeared to be comparable. In RA patients, after multipleintravenous infusions, the pharmacokinetics of abatacept showedproportional increases of C_(max) and AUC over the dose range of 2 mg/kgto 10 mg/kg. At 10 mg/kg, serum concentration appeared to reach asteady-state by day 60 with a mean (range) trough concentration of 24mcg/mL (from about 1 to about 66 mcg/mL). No systemic accumulation ofabatacept occurred upon continued repeated treatment with 10 mg/kg atmonthly intervals in RA patients.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All citations throughout the disclosure arehereby expressly incorporated by reference.

Example I

Belatacept SC, 125 mg/ml (100 mg/vial) drug product is formulated as asterile, non-pyrogenic ready-to-use solution suitable for subcutaneousadministration.

Belatacept SC drug product, 125 mg/ml (100 mg/vial) is manufactured at2.2 kg scale (1500 vials). The batch formula is provided in Table 7below.

TABLE 7 Representative Batch Formula Component Amount Per Batch (g)Belatacept drug substance^(b) 250.0 Sucrose 340.0 Poloxamer 188 16.0Sodium phosphate monobasic, monohydrate 0.662 Sodium phosphate dibasic,anhydrous 2.13 Water for Injection q.s. to 2.2 kg Total Batch Size (kg)2.2 kg ^(a) ^(a) Total batch size 2.2 kg (2-L). The density of thesolution is 1.10 g/ml (at 20°-25° C.) ^(b)Belatacept drug substance:protein concentration 25 mg/ml, 25 mM sodium phosphate, 10 mM sodiumchloride, pH of 7.5, <5% HMW species

The manufacturing process for Belatacept SC drug product, 125 mg/ml (100mg/vial) drug product involves buffer exchange of the bulk drugsubstance from 25 mM sodium phosphate, 10 mM sodium chloride buffer at apH of 7.5 to 10 mM sodium phosphate pH 7.5 buffer, followed byconcentration of the protein from ˜25 mg/ml to ˜150 mg/ml by removal ofbuffer. The buffer exchange is accomplished by five times diafiltrationof the bulk drug substance against the new 10 mM sodium phosphate pH 7.5buffer, followed by concentration of protein to ˜150 mg/ml by removal ofbuffer. A stainless steel Pelicon mini filter holder (Millipore) isequipped with stainless steel pressure gauges and membrane valves on thefeed, retentate and permeates port. Two filtration cassettes used withPellicon mini module are fitted with 0.1 m² area Biomax polyethersulfonemembrane with 30 kDa nominal molecular weight cutoff limit. Thefiltration cassettes are installed according to the manufacturer'srecommendations. The feed container for the drug substance is a 4 literglass container with magnetic stir bar. MasterFlex high performancesilicone tubing is used to connect the feed container to the filterholder and for the permeate line. Feed flow is provided by peristalticpump installed in the feed line. Sucrose and Poloxamer 188 are thendissolved in the concentrated protein solution and final batch weight isadjusted with 10 mM sodium phosphate buffer, pH 7.5. The bulk solutionis filtered through 0.22 micron sterilizing filter and filled intosterilized and depyrogenated 5-cc Type I flint glass vials, stopperedwith 20 mm rubber stoppers and sealed with 20 mm aluminum flip-offseals. The composition of Belatacept SC drug product, 125 mg/ml (100mg/vial) drug product is provided in Table 8 below.

TABLE 8 Composition of Belatacept SC drug product, 125 mg/ml (100mg/vial) Component Amount (mg/vial)^(d) Belatacept 140.0 Sucrose 190.4Poloxamer 188 8.96 Sodium phosphate monobasic, monohydrate 0.371 Sodiumphosphate dibasic, anhydrous 1.193 Water for Injection q.s. to 1.12 ml^(d)Includes 40% overfill for Vial, Needle, Syringe loss

Example II

Abatacept SC, 125 mg/ml (125 mg/vial) drug product is formulated as asterile, non-pyrogenic ready-to-use solution suitable for subcutaneousadministration. A batch of Abatacept SC, 125 mg/ml (125 mg/vial) drugproduct is manufactured at 5-L scale (3,500 vials). The batch formula isdescribed in Table 9 below.

TABLE 9 Batch Formula Component Amount (gm) Abatacept drug substance^(a)625 Sucrose 850 Poloxamer 188 40 Sodium phosphate monobasic, monohydrate0.715 Sodium phosphate dibasic, anhydrous 4.86 Water for Injection q.s.to 5.0 L Total Batch size (L) 5.0 ^(a)Abatacept drug substance: proteinconcentration 50 mg/ml, 25 mM sodium phosphate, 50 mM sodium chloride,pH of 7.5, <5% HMW species

As described above in Example I, the manufacturing process for AbataceptSC, 125 mg/ml (125 mg/vial) drug product involves buffer exchange of thebulk drug substance from 25 mM sodium phosphate, 50 mM sodium chlorideat a pH of 7.5 to 10 mM sodium phosphate pH 7.8 buffer, followed byconcentration of the protein from ˜50 mg/ml to ˜150 mg/ml by removal ofbuffer. Sucrose and Poloxamer 188 are then dissolved in the concentratedprotein solution and final batch weight is adjusted with 10 mM sodiumphosphate buffer, pH 7.8. The bulk solution is filtered through 0.22micron sterilizing filter and filled into sterilized and depyrogenated5-cc Type I flint glass vials, stoppered with 20 mm rubber stoppers andsealed with 20 mm aluminum flip-off seals.

The composition of Abatacept SC drug product, 125 mg/ml (125 mg/vial) isprovided in Table 10 below.

TABLE 10 Composition of Abatacept SC, 125 mg/ml (125 mg/vial) drugproduct Component Amount (mg/vial)^(e) Abatacept 175 Sucrose 238Poloxamer 188 11.2 Sodium phosphate monobasic, monohydrate 0.20 Sodiumphosphate dibasic, anhydrous 1.36 Water for Injection q.s. to 1.4 ml^(d) Includes 40% overfill for Vial, Needle, Syringe loss

Example III

Abatacept, lyophilized, (250 mg/vial) drug product is a sterile,non-pyrogenic lyophile suitable for intravenous (IV) administration.Each single-use vial contains 250 mg of abatacept which is constitutedwith Sterile Water for Injection, USP and further diluted with 0.9%Sodium Chloride Injection, USP, at the time of use.

The batch formula for a 115 liter batch size is described in Table 11below.

TABLE 11 Batch formula Component Amount (kg) Abatacept drugsubstance^(a) 4.6 Maltose monohydrate 9.2 Hydrochloric Acid Adjust to pH7.5 Sodium hydroxide Adjust to pH 7.5 Water for Injection q.s. to119.6^(b) ^(a)abatacept drug substance: protein concentration 50 mg/ml,25 mM sodium phosphate, 50 mM sodium chloride, pH of 7.5, <5% HMWspecies ^(b)formulated bulk solution density = approx. 1.04 g/ml

The required quantity of abatacept drug substance is added to a cleanedand sterilized stainless steel compounding vessel equipped with a mixer.The drug substance solution is mixed at 250±50 rpm while maintaining thesolution temperature between 5°-25° C.

The required quantity of maltose monohydrate powder is added to thecompounding vessel. The solution is mixed for a minimum of 10 minutes at15°-25° C.

The solution pH is adjusted to 7.3-7.7, if necessary using thepreviously prepared 1 N sodium hydroxide solution or 1 N hydrochloricacid solution. The batch is brought to the final batch weight (finalq.s.) using Water for Injection, USP, and mixed for a minimum of 8minutes. The formulated bulk solution is sampled for pH.

Formulated Bulk Solution is pre-filtered with one 0.45-μm filter. Theformulated bulk solution after 0.45-μm filter is sampled for bioburdenand bacterial endotoxin (BET).

The pre-filtered formulated bulk Solution is sterile filtered with two0.22-μm filters in series prior to filling.

Sterile filtered Formulated Bulk Solution is filled and partiallystoppered with a 20 nm-Daikyo gray butyl stopper by a fully automaticfilling/stoppering machine. The 15-cc Type I flint tubing glass vialsare washed and sterilized/depyrogenated.

The filled and partially stoppered drug product vials are lyophilized. Asummary of the freeze drying cycle used during lyophilization ofabatacept drug product is provided in Table 12 below.

TABLE 12 Freeze dry cycle for abatacept lyophilized drug product Processparameter In-process control Loading Temperature 5 ± 3° C. Freezing(Shelf Ramp) From 5° C. to −45° C. in 2.5 hours Freezing Hold at −45 ±3° C. for 4 hours Primary Drying (Shelf Ramp) From −45° C. to −19° C. in2 hours Primary Drying (Vacuum) 100 ± 20 microns Primary Drying Hold at−19 ± 2° C. for 84 hours Intermediate Drying From −19° C. to 0° C. in 2hours (Shelf Ramp) Intermediate Drying Hold at 0 ± 3° C. for 8 hoursSecondary Drying (Shelf Ramp) From 0° C. to 30° C. in 2.5 hoursSecondary Drying (Vacuum) 100 ± 20 microns Secondary Drying Hold at 30°C. for 12 hours Stoppering 30 ± 3° C. Stoppering (Vacuum) 500 ± 100microns Storage Before Unloading Hold at 20 ± 3° C. for at least 4 hours

At the end of the lyophilization cycle, the chamber pressure is raisedto 500 microns using sterile filtered nitrogen and vial stoppering isperformed under vacuum. The stoppered vials remain inside thelyophilizer for at least 4 hours. The lyophilized and stoppered vialsare sealed with a 20-mm aluminum, white flip-off seal under HEPAfiltered air by the capping machine. The sealed vials are rinsed withdeionized water by an exterior vial washer. The washed drug productvials are stored at 2 to 8° C.

The composition of lyophilized abatacept (250 mg/vial) drug product islisted in Table 13 below.

TABLE 13 Composition of lyophilized abatacept (250 mg/vial) drug productComponent Amount (mg/vial)^(f) Abatacept 262.5 Maltose monohydrate 525Sodium phosphate monobasic, monohydrate^(b) 18.1 Sodium chloride^(b)15.3 Hydrochloric Acid Adjust to 7.5 Sodium hydroxide Adjust to 7.5 ^(a)includes a 5% overfill for vial, needle, syringe loss ^(b)Thesecomponents are present in the abatacept drag substance solution

Example IV

Belatacept, lyophilized, (100 mg/vial), drug product is a sterile,non-pyrogenic lyophile suitable for intravenous (IV) administration.Each single-use vial contains 100 mg of belatacept with is constitutedwith 4.2 ml of Sterile Water for Injection, USP to yield a concentrationof 25 mg/ml. It can be further diluted to a concentration as low as 1mg/ml with 5% Dextrose Injection, USP or 0.9% Sodium Chloride Injection,USP at the time of use.

The batch size for drug product manufacture may vary from 20 liters to120 liters. A representative batch formula for a batch size of 66 liters(12,000 vials) is provided in Table 14 below.

TABLE 14 Batch Formula for a Batch Size of 66 liters (12,000 vials)Component Amount (kg) Belatacept drug substance^(a) 1.32 Sucrose, HighPurity, Low Endotoxin 2.64 Sodium Phosphate Monobasic Monohydrate 0.18Sodium Chloride 0.03 1N Sodium Hydroxide/or 1N Hydrochloric Acid AdjustpH to 7.5 Water for Injection q.s. to batch weight ^(a)Belatacept drugsubstance: protein concentration 25 mg/ml, 25 mM sodium phosphate, 10 mMsodium chloride, pH of 7.5, <5% HMW species

Belatacept lyophilized drug product is manufactured as described inExample III above.

The composition of lyophilized belatacept drug product, 100 mg/vial islisted in Table 15 below.

TABLE 15 Composition of lyophilized belatacept 100 mg/vial drug productComponent Amount/Vial (mg) Belatacept 110^(a)   Sucrose, High Purity,Low Endotoxin 220    Sodium Phosphate Monobasic Monohydrate 15.18 SodiumChloride  2.55 1N Sodium Hydroxide Adjust to pH 7.5 1N Hydrochloric AcidAdjust to pH 7.5 ^(a)Each vial contains 10% overfill for vial, needleand syringe holdup of the reconstituted solution.

Example V

Stability studies of the SC liquid formulation of Belatacept drugproduct were conducted by placing formulations on stability at differenttemperatures and for various time periods.

Effect of Sucrose

Formulation development studies were conducted to evaluate the effect ofvarious levels of sucrose on solution stability of belatacept drugproduct. Samples were placed on stability at −70° C., 8° C. and 25°C./60% humidity conditions and monitored at various time points. Theratios of protein to sucrose evaluated were 1:1, 1:1.7 and 1:1.75. Theformation of high molecular weight (HMW) species of belatacept wasutilized to determine protein stability in solution. Results are shownin Table 16 below.

TABLE 16 Effect of various levels of sucrose on Belatacept drug productat 100 mg/ml Time/ % High Molecular Weight Species Storage ConditionMonths Control ^(a) 1:1 1:1.75 1:1.7 Initial 0 1.11 1.07 1.05 1.06 −70°C. 3 1.18 1.09 1.07 1.09  8° C. 1 1.22 1.10 1.08 1.07 2 1.34 1.15 1.081.09 3 NA 1.24 1.15 1.17 6 1.78 1.39 1.23 1.24 9 2.02 1.53 1.34 1.34 122.06 1.49 1.26 1.24 25° C./60% RH 0 1.11 1.07 1.05 1.06 1 2.73 1.83 1.481.56 2 3.98 2.41 1.84 1.87 3 4.88 3.02 NA 2.23 6 7.44 4.56 3.21 NA ^(a)Belatacept drug product in 10 mM sodium phosphate buffer, 100 mg/ml, pH7.5

The results of the studies showed that increasing the sucrose to proteinratio improved protein stability. A protein to sucrose ratio of 1:1.36(wt.:wt.) was chosen for the development of the SC solution because itprovided optimum stability without resulting in drug product withexcessive hypertonicity.

Effect of Surfactants

The effect of various surfactants in marketed products, such asPolysorbate 80 and Poloxamer 188 on the solution stability of belataceptdrug product was evaluated. Poloxamer 188 was evaluated at levels of 4,6 and 8 mg/ml and Polysorbate 80 was evaluated at 1 and 2 mg/ml of finalformulation concentration. Samples were placed on stability at −70° C.,8° C. and 25° C./60% humidity conditions and monitored at various timepoints. Results are shown in Table 17 below.

TABLE 17 Effect of various levels and types of surfactant on Belataceptdrug product (100 mg/ml) % High Molecular Weight Species Poloxamer 188Polysorbate Time/ mg/ml 80 mg/ml Storage Condition Months Control^(a) 46 8 1 2 Initial 0 1.11 1.06 1.07 1.07 1.08 1.08 −70° C. 3 1.18 1.09 NA1.09 1.11 1.15  8° C. 1 1.22 1.10 1.08 1.09 1.10 1.12 2 1.34 1.09 1.101.11 1.12 1.12 3 NA 1.19 1.18 1.18 1.21 1.28 6 1.78 1.27 1.23 1.25 1.291.30 9 2.02 1.34 1.33 1.34 1.42 1.40 12 2.06 1.28 1.25 1.27 1.38 1.3625° C./60% RH 0 1.11 1.06 1.07 1.07 1.08 1.08 1 2.73 1.52 1.52 1.52 1.551.55 2 3.98 1.91 1.89 1.89 1.99 1.97 3 4.88 2.31 2.29 2.24 2.56 2.50 67.44 3.39 4.05 NA 3.89 3.90 ^(a)protein:sucrose (1:1.7), 100 mg/ml, pH7.5

Results of the effect of surfactants suggested that surfactant did nothave a significant effect on the stability of belatacept drug productsolution. Among the levels of Poloxamer 188 evaluated. the concentrationof 8 mg/ml was found to be adequate to prevent the formation of siliconerelated particulates in the formulation.

Effect of pH

Stability of the Belatacept SC, (125 mg/ml, protein:sucrose 1:1.36, 8mg/ml Pluronic F68) drug product was evaluated as a function of pH. Thesolution pH was adjusted between 7 to 8.2 with either 1N sodiumhydroxide or 1N Hydrochloric acid. Samples were placed on stability at2°-8° C. and 25° C./60% RH conditions and monitored at various timepoints. Analytical testing included pH and SE-HPLC to monitor increasein high molecular weight (HMW) species. These results are summarized inTable 18 below.

TABLE 18 Effect of pH on Belatacept SC drug product* Time/ % HighMolecular Weight Species Condition Months pH 7.0 pH 7.4 pH 7.8 pH 8.2Initial 0 1.31 1.18 1.16 1.28 2-8° C. 1 1.34 1.23 1.25 1.44 2 1.42 1.293.31 1.56 3 1.48 1.35 1.36 1.59 25° C./60%RH 1 2.09 2.62 2.13 2.52 27.04 5.68 5.89 6.40 3 9.98 6.13 NA 7.81 *protein:sucrose (1:1.36), 125mg/ml, +8 mg/ml Pluronic F68

No significant changes in the rate of formation of HMW species wereobserved under the recommended storage condition of 2-8° C.Additionally, the solution-state stability data showed the pH of maximumstability to be between 7 and 8. Based on this, a pH range of 7-8 with atarget pH of 7.5 was selected for this formulation.

Osmolality

Osmolality of belatacept drug product solutions in various buffers, atdifferent protein concentrations and from separate steps of theformulation process were measured using a vapor pressure method. Theseresults are summarized in Table 19 below.

TABLE 19 Osmolality Determination of Belatacept Drug Product Solution inVarious Buffer and Concentration protein Sodium Osmo- Belatacept/Buffer/Conc Phos- Sodium lality Excipients (mg/ml) phate Chloride (mOsm/kg)Belatacept API, as is 25 25 mM 10 mM 89 Belatacept, Diafiltration 25 10mM — 40 step Belatacept Diafiltration/ 138 10 mM — 58 Concentration stepBelatacept in water 25 — — 17 Belatacept in water 100 — — 27Belatacept:Sucrose (1:1) 100 10 mM — 424 Belatacept:Sucrose (1:1.7) 10010 mM — 737 Belatacept:Sucrose (1:1.75) 100 10 mM — 769Belatacept:Sucrose (1:2) 100 10 mM — 870 Belatacept:Sucrose (1:1.36) 12510 mM — 770

Effect of Agitation/Shaking

The effect of agitation on solution stability of belatacept SC drugproduct at 100 mg/ml and 125 mg/ml concentration was determined.Aliquots of the solution containing approximately 1 ml in 5 cc tubingvials were shaken at speed 3 of wrist arm shaker at 2-8° C. Thetemperature of the shaker was maintained at 2-8° C. by placing theshaker in the cold room. Samples were withdrawn at appropriate timeintervals and assayed for pH and visual appearance, and same timesamples were also evaluated for bioactivity after 30 days of agitation.

Samples agitated at 100 mg/ml and 125 mg/ml concentration for up to 30days show no change in the level of HMW species, in SDS-PAGE profile,peptide mapping, B7 binding assay, pH, appearance or proteinconcentration when agitated at 2-8° C.

Effect of Multiple Freeze/Thaw

The effect of multiple freezing and thawing on stability of belataceptSC drug product formulation was investigated in samples with pH rangingfrom 7.0 to 8.2. Approximately 10 μl aliquots of belatacept SC drugproduct formulation (125 mg/ml) at pH 7.0, 7.4, 7.8 and 8.2 weredispensed into 30 ml Nalgene PETG containers. Multiple freezing andthawing were performed by storing vials at −70° C. followed by thawingat ambient temperature (25° C.) for 10 minutes. This cycle was repeatedfor 5 days. The contents of vials were analyzed for pH, % HMW speciesand appearance after each freeze/thaw cycle.

No change in pH, appearance or % high molecular weight species contentwas observed in samples during five freeze/thaw cycles.

Recommended Storage Conditions

The recommended storage condition for Belatacept SC drug product, 100mg/vial (125 mg/ml) is 2-8° C. with a recommended shelf life of 12months.

Syringe-Ability Study

Syringe-ability study was performed with belatacept SC drug product (125mg/ml) at 2°-8° C. condition. Various needle sizes with 1 ml and 0.5 mLsyringe were evaluated. Syringe filling time and delivery force arerecorded in Table 20 below.

TABLE 20 Belatacept SC drug product, 125 mg/ml Syringeability Study at2°-8° C. Needle Filling Time Delivery Syringe Size size (Sec) Force 1 mL27G ½″ 25 Moderate 1 mL staked needle (Insulin) 28G ½″ 52 Moderate 1 mLstaked needle (Insulin) 29G ½″ 50 Moderate 0.5 mL staked needle(Insulin) 30G 42 (94 for 1 mL) High

Based on the syringe-ability study results shown in Table 20. A 21gauge×1½ inch sterile hypodermic needle is recommended for withdrawal ofthis product from vial and a 27 gauge×½ inch needle for subsequentdosing.

Example VI

Stability studies of the lyophilized formulation of Abatacept drugproduct were conducted by placing formulations on stability at differenttemperatures and for various time periods.

Effect of Maltose

Formulation development studies were conducted to evaluate the effect ofvarious levels of maltose on the stability of abatacept drug product.Samples were placed on stability at 50° C. and monitored at various timepoints. The ratios of protein to maltose evaluated were 1:1, 1:2 and1:5. The formation of high molecular weight (HMW) species of abataceptwas utilized to determine protein stability in solid state. Results areshown in Table 21 below.

TABLE 21 Effect of Maltose on the Freeze-Dried Solid-State Stability ofAbatacept drug product at 50° C. Level of High Molecular Weight Speciesby SE-HPLC (Area %) Drug to Maltose Weight Ratio Time (Weeks) WithoutMaltose 1:1 1:2 1:5^(a) Initial 0.9 0.7 0.6 2.0 2 5.4 3.7 2.0 NA 4 8.05.9 2.7 2.4 6 10.2 6.8 3.3 NA 8 11.7 7.5 3.9 2.9 ^(a)Stability of drugproduct with 1:5 drug-to-maltose weight ratio was evaluated during earlydevelopment with 50 mg/vial strength. The drug substance lot used inthis study was different from that used for other results in this table.This is the reason for the different initial levels of high molecularweight species in these samples.

The results demonstrate that the stability of abatacept drug product ina freeze-dried solid-state form is enhanced in the presence of maltose.Additionally, the minimum amount of maltose useful for stabilization ofabatacept was determined to be at a 1:2 protein to maltose weight ratio.

Effect of pH

Stability of lyophilized Abatacept drug product, (250 mg/vial,protein:maltose 1:2) was evaluated as a function of pH. The solution pHwas adjusted between 6 to 8 with either 1N sodium hydroxide or 1NHydrochloric acid. Samples were placed on stability at 2°-8° C.conditions and monitored at various time points. Analytical testingincluded pH and SE-HPLC to monitor increase in high molecular weight(HMW) species. These results are summarized in Table 22 below.

TABLE 22 Effect of pH on the Rate of Formation of High Molecular Weight(HMW) Species HMW Species Level (Area %) by SE-HPLC at Various pH ValuesTime (months) 6.0^(a) 6.5^(a) 7.0 7.5 8.0 Initial 0.6 0.6 2.0 2.0 2.0 10.5 0.6 2.0 2.0 2.0 2 0.7 0.7 2.0 2.0 2.0 3 0.7 0.7 2.1 2.1 2.1 6 NA NA1.8 1.7 1.8 12  0.8 0.8 1.9 1.8 1.9 ^(a)The drag substance lot used forpH 6.0 and 6.5 samples was different from that used for pH 7.0. 7.5 and8.0 samples. This is the reason for the different initial levels of highmolecular weight species in these samples.

Under the recommended storage condition of 2°-8° C., no significantchanges in the rate of formation of HMW species were observed.Additionally; the solution-state stability data generated during anearly development showed the pH of maximum stability to be between 7 and8.

Example VII

Stability studies of the liquid formulation of belatacept (20 mg/ml)drug product were conducted by placing formulations on stability atdifferent temperatures and for various time periods.

Effect of Sucrose

The formulation development studies were conducted to evaluate theeffect of various levels of sucrose on the solution stability ofbelatacept liquid drug product at 20 mg/ml. Samples were placed onstability at 8° C., 25° C./60% humidity and 30° C./60% humidityconditions and monitored at various time points. The ratios of proteinto sucrose evaluated were 1:1, 1:2, 1:5 and 1:10 protein:sucrose ratiowith 20 mg/mL of belatacept. The formation of high molecular weight(HMW) species of belatacept was utilized to determine protein stabilityin solution. Results of these study is summarized and shown in Table 23below.

TABLE 23 Effect of various levels of sucrose on liquid belatacept drugproduct, 20 mg/ml % High Molecular Time/ Weight Species Condition Months1:1 1:2 1:5 1:10 Initial 0 0.37 0.37 0.39 0.37 8° C. 1 0.39 0.39 0.380.36 2 0.40 0.41 0.38 0.37 3 0.43 0.39 0.36 0.36 4 0.44 0.42 0.40 0.37 60.58 0.47 0.41 0.39 9 0.54 0.45 0.42 0.39 12 0.52 0.45 0.44 0.51 25°C./60% RH 1 0.61 0.62 0.66 0.50 2 1.25 0.99 0.58 0.53 3 1.40 1.00 0.800.57 4 1.72 1.40 1.10 0.74 6 4.29 2.70 2.09 1.18 9 —* 5.59 4.15 2.33 30°C./60% RH 0.5 1.13 0.91 0.75 0.54 1 1.77 2.03 1.25 0.84 2 3.01 2.90 1.781.10 3 4.32 11.24 5.55 1.75 *Samples not available for analysis due toevaporation

The results of the studies showed that increasing the sucrose to proteinratio improved protein stability. A protein to sucrose ratio of 1:2(wt.:wt.) was chosen for the development of the liquid solution becauseit provided optimum stability without resulting in drug product withexcessive hypertonicity.

Stability Study

Three liquid beleatacept batches were prepared and placed on stabilityat 2-8° C. and 25° C./60% RH conditions and monitored at various timepoints. The weight ratio of protein to sucrose was 1:2. The formation ofhigh molecular weight (HMW) species of belatacept was utilized todetermine protein stability in liquid formulation. Stability data issummarized in Table 24 below.

TABLE 24 Stability of liquid belatacept drug product Belatacept drugProtein:Sucrose (1:2), substance 20 mg/mL pH 7.5 Time (25 mg/mL) % HMWspecies Condition Months Control Batch 1 Batch 2 Batch 3 Initial 0 0.400.37 0.43 0.91 5° C. 1 0.44 0.39 0.45 0.90 2 0.47 0.41 0.46 0.90 3 0.470.39 0.62 0.91 4 0.54 0.42 NA 0.92 6 0.63 0.47 0.57 0.94 9 0.64 0.450.51 1.0  12 0.64 0.45 0.56 NA 25° C./60% RH 1 0.61 0.62 0.60 1.04 21.25 0.99 0.90 1.23 3 1.40 1.00 1.18 1.53 4 1.72 1.40 1.60 1.79 6 4.292.70 3.09 2.44

The data indicate only 0.1% increase in high molecular weight species inliquid formulation compared to 0.2% increase in Belatacept drugsubstance without sucrose in 12 months at 2-8° C. These results alsoindicate that addtion of sucrose does help in reducing formation of highmolecular weight species.

Example VIII

Stability studies of the SC formulation of Abatacept drug product wereconducted by placing formulations on stability at different temperaturesand for various time periods.

Effect of Buffer Strength

Stability of SC Abatacept drug product, (100 mg/ml) was evaluated as afunction of buffer strength. The buffer system was either 5 or 10 mMphosphate buffer. Samples were placed on stability at 2°-8° C. and 30°C./60% RH conditions and monitored at various time points. Analyticaltesting included pH and SE-HPLC to monitor increase in high molecularweight (HMW) species. These results are summarized in Table 25 below.

TABLE 25 Effect of buffer strength on Abatacept drug product (100 mg/ml,pH 7.5) % High Molecular Weight Species Time/ In 10 mM phosphate In 5 mMphosphate Storage Condition Months buffer buffer Initial 0 1.30 1.472-8° C. 1 1.49 1.74 2 1.60 1.98 3 1.73 2.23 30° C./60% RH 0 1.30 1.470.5 8.73 14.39 1 14.63 23.24 2 25.26 32.25

Stability of the abatacept SC drug product was better in 10 mM phosphatebuffer compared to 5 mM phosphate buffer at pH 7.5 at 100 mg/mLabatacept concentration. Moreover the higher buffering capacity of 10 mMphosphate buffer offered better pH control of the formulation comparedto 5 mM buffer. Based on the data, 10 mM phosphate buffer was selectedfor formulation development.

Effect of Sugars

Formulation development studies were conducted to evaluate the effect ofvarious sugars on solution stability of abatacept SC drug product.Samples were placed on stability at 2-8° C. and 30° C./60% humidityconditions and monitored at various time points. The sugars evaluatedwere sucrose, trehalose and mannitol. The formation of high molecularweight (HMW) species of abatacept was utilized to determine proteinstability in solution. Results are shown in Table 26 below.

TABLE 26 Effect of various sugars on Abatacept SC drug product at 100mg/ml, pH 7.5 % High Molecular Weight Species Time/ 1:1 1:1 1:1 StorageCondition Months Control^(a) Sucrose Trehalose Mannitol^(b) Initial 01.30 1.18 1.20 1.20 2-8° C. 1 1.49 1.37 1.38 1.25 2 1.60 1.40 1.41 1.263 1.73 1.45 1.48 1.60 6 2.10 1.54 N/A N/A 11.5 2.57 N/A N/A N/A 30°C./60% RH 0 1.30 1.18 1.20 1.30 0.5 8.73 4.31 4.34 3.59 1 14.63 7.208.09 5.72 2 25.26 11.97 14.21 10.14 ^(a)Abatacept drug product in 10 mMsodium phosphate buffer, 100 mg/ml, pH 7.5. ^(b)Mannitol formulation atpH 7.8

The results of the studies showed that all three sugars sucrose,trehalose and mannitol offered better stabilization to abataceptcompared to the control without sugar. The results of the studies underaccelerated conditions of 30 C showed that mannitol offered betterstabilization to abatacept compared to sucrose and trehalose. Sucrosewas slightly better than trehalose. Under refrigeration, thestabilization by all three sugards was not significantly different.Sucrose was chosen as sugar of choice since mannitol formulation hadtwice the tonicity of the sucrose formulation. Choosing sucrose forstabilization would allow addition of twice as much sucrose to achievethe same tonicity as mannitol at the same ratio but much greaterstabilization against aggregation. A protein:sucrose in ratio of 1:1.36(wt.:wt.) was chosen for the development of the SC drug product becauseit provided optimum stability without resulting in a drug product withexcessive hypertonicity.

Effect of Sucrose

Formulation development studies were conducted to evaluate the effect ofvarious levels of sucrose on solution stability of abatacept SC drugproduct. Samples were placed on stability at 2-8° C. and 30° C./60%humidity conditions and monitored at various time points. The ratios ofprotein to sucrose evaluated were 1:1 and 1:2. The formation of highmolecular weight (HMW) species of abatacept was utilized to determineprotein stability in solution. Results are shown in Table 27 below.

TABLE 27 Effect of various levels of sucrose on Abatacept SC drugproduct at 100 mg/ml, pH 7.5 % High Molecular Time/ Weight SpeciesStorage Condition Months Control^(a) 1:1 1:2 Initial 0 1.30 1.18 1.172-8° C. 1 1.49 1.37 1.33 2 1.60 1.40 1.21 3 1.73 1.45 1.23 6 2.10 1.541.29 11.5 2.57 N/A N/A 30° C./60% RH 0 1.30 1.18 1.17 0.5 8.73 4.31 2.411 14.63 7.20 3.69 2 25.26 11.97 6.59 ^(a)Abatacept drug product in 10 mMsodium phosphate buffer, 100 mg/ml, pH 7.5

The results of the studies showed that increasing the sucrose to proteinratio improved protein stability. A protein:sucrose ratio of 1:1.36(wt.:wt.) was chosen for the development of the RTU solution because itprovided optimum stability without resulting in drug product withexcessive hypertonicity.

Effect of Surfactants

The effect of various surfactants in marketed products, such asPolysorbate 80 (Tween® 80) and Poloxamer 188(Pluronic® F68) on thesolution stability of abatacept SC drug product was evaluated. Poloxamer188 was evaluated at levels of 4 and 8 mg/ml and Polysorbate 80 wasevaluated at 1 and 2 mg/ml of final formulation concentration. Sampleswere placed on stability at −2-8° C. and 25° C./60% humidity conditionsand monitored at various time points. Results are shown in Table 28below.

TABLE 28 Effect of various levels and types of surfactant on abataceptSC drug product at 125 mg/ml % High Molecular Weight Species PoloxamerPolysorbate Time/ 188 mg/ml 80 mg/ml Storage Condition MonthsControl^(a) 4 8 1 2 Initial 0 1.05 1.05 1.06 1.07 1.10 2-8° C. 1 0.980.99 0.99 1.01 1.02 2 1.02 1.03 1.04 1.05 1.07 3 1.06 1.07 1.08 1.081.09 6 1.23 1.27 1.28 1.25 1.32 9 1.30 1.35 1.36 1.34 1.36 25° C./60% RH0 1.05 1.05 1.06 1.03 1.04 1 1.92 1.94 1.92 1.97 2.02 2 2.69 2.71 2.682.75 2.80 3 3.74 3.92 3.67 3.84 3.84 5 5.12 5.16 5.03 5.05 5.03^(a)protein:sucrose (1:1), 125 mg/ml, pH 7.8

Results of the effect of surfactants suggested that surfactant did nothave a significant negative effect on the stability of abatacept SC drugproduct. Among the levels of Poloxamer 188 evaluated. the concentrationof 8 mg/ml was found to be adequate to prevent the formation of siliconerelated particulates in the formulation.

Osmolality

Osmolality of abatacept in various buffers, at different proteinconcentrations and from separate steps of the formulation process weremeasured using a vapor pressure method. These results are summarized inTable 29 below.

TABLE 29 Osmolality Determination of Abatacept SC Solution in VariousBuffer and Concentration proteinConc Sodium Sodium OsmolalityAbatacept/Buffer/Excipients (mg/ml) Phosphate Chloride (mOsm/kg)Abatacept drug substance 50 25 mM 50 mM 151 Abatacept, Diafiltrationstep 50 10 mM — 32 Abatacept Diafiltration/ 155 10 mM — 49 Concentrationstep 10 mM pH 7.8 phosphate buffer — 10 — 35 Abatacept in water 100 10mM — 51 Abatacept:Sucrose (1:1) 100 10 mM — 567 Abatacept:Sucrose(1:1.75) 100 10 mM — 766 Abatacept:Sucrose (1:2) 100 10 mM — 913Abatacept:Sucrose (1:1.36) 125 10 mM — 782

Effect of Agitation/Shaking

The effect of agitation on solution stability of abatacept SC drugproduct at 100 mg/ml and 125 mg/ml concentration was determined.Aliquots of the solution containing approximately 1 ml in 5 cc tubingvials were shaken at speed 3 of wrist arm shaker at 2-8° C. Thetemperature of the shaker was maintained at 2-8° C. by placing theshaker in the cold room. Samples were withdrawn at appropriate timeintervals and assayed for pH and visual appearance, and same timesamples were also evaluated for bioactivity after 30 days of agitation.

Samples agitated at 100 mg/ml and 125 mg/ml concentration for up to 30days show no change in the level of HMW species, in SDS-PAGE profile,peptide mapping, B7 binding assay, pH, appearance or proteinconcentration when agitated at 2-8° C.

Recommended Storage Conditions

The recommended storage condition for abatacept SC drug product, 125mg/Syringe (125 mg/ml) is 2-8° C. with a recommended shelf life of atleast 12 months.

Example IX

Deamidation and aggregation are two observed degradation pathways ofCTLA4Ig molecules. This protocol outlines a laboratory scale pHstability study designed to evaluate the SC drug product formulation inthe pH range of 6.3-7.2, specifically pH 6.3, 6.6, 6.9, 7.2. The purposeof this study is to identify the optimal lower pH formulation that willattain a minimum of 18-months of shelf-life for the CTLA4Ig SCformulations with regards to deamidation and formation of high molecularweight species. The SC drug product formulation utilized in this studyis described in Table 30 below.

TABLE 30 SC drug product formulation composition at pH 6.9 IngredientAmount mg/1.0 ml abatacept 125 Sucrose 170 Poloxamer 188 8 Monobasicsodium phosphate, monohydrate 0.638 Dibasic sodium phosphate, anhydrous0.475 Water for Injection QS 1.0 mL

Abatacept SC drug product will be formulated at pH 6.3, 6.6, 6.9, 7.2.The drug product will be formulated with sucrose and poloxamer 188 asdescribed above and the final batch concentration will be adjusted with10 mM phosphate buffer (pH 6.9). The pH will be titrated down to 6.3 and6.6, respectively, using 1N HCl. Alternatively, the pH will be titratedup to 6.9, 7.2, and 7.65 with 1N NaOH. The drug product will be filledinto 1-mL long Physiolis™ syringes (1.0 ml fill volume) and placed onstability stations at 2-8° C., 15° C., 25° C. at 60% humidity, and 35°C. Samples should be protected from light at all times by covering orinserting into brown light-protective bags.

Drug product stored at 2-8° C. will be sampled at 0, 2, 4, 6, 12 18, 24months and optionally 9 months. Drug product stored at 15° C. will besampled at 1, 2, 4, 6 months and optionally 9 months. Drug productstored at 25° C. and 60% humidity will be sampled at 1, 2, 4 and 6months. Drug product stored at 35° C. will be sampled at 1, 2 and 4months. Samples stored at 2-8° C. will be tested for appearance (initialand last samples only), pH (initial, 4 month and last samples only),A280(initial, 4 month and last samples only), size exclusion HPLC,SDS-PAGE, tryptic digest peptide mapping (TPM), Biacore B7 Binding(initial, 4 month and last samples only or as needed) and isoelectricfocusing(IEF)(initial and last samples only). Samples stored at 15° C.and 25° C. and 60% humidity will be tested for A280(initial, 4 monthsand last samples only), size exclusion HPLC, SDS-PAGE and tryptic digestpeptide mapping (TPM). Samples stored at 35° C. will be tested for sizeexclusion HPLC, SDA-PAGE and tryptic digest peptide mapping (TPM).

Non-Routine testing methods will be used to further characterizestability samples at the initial time point, 4 months, 12 months and atthe end of the study. Some of these methods may also be used to testspecific samples if a trend or an unexpected result is observed. Thenon-routine methods include: size exclusion chromatography employingmultiangle light scattering (SEC-MALS), kinetic binding (SPR), MassSpectrometry, CD, AUC, Differential Scanning calorimeter (DSC), FFF,FTIR, size exclusion HPLC (denatured) and SDS-PAGE (silver stain).

Example X

A PK substudy was incorporated in a phase 2B, multi-center, randomized,double-blind, placebo-controlled study to evaluate the safety andclinical efficacy of two different doses of abatacept administeredintravenously to subjects with active rheumatoid arthritis whilereceiving methotrexate. In this parallel design study, subjects receivedabatacept at 2 different doses (2 and 10 mg/kg) or placebo incombination with MTX. Abatacept was manufactured as described inco-pending U.S. patent application Ser. No. 60/752,267, filled on Dec.20, 2005 which teaches processes for the production of proteins of theinvention, specifically recombinant glycoprotein products, by animal ormammalian cell cultures, and supplied in lyophilized form, as describedherein, in individual vials containing 200 mg of abatacept. Abataceptwas administered IV to subjects on Days 1, 15, and 30, and every 30 daysthereafter for a year. Multiple dose PK was derived from the serumconcentration vs time data obtained during the dosing interval betweenDays 60 and 90 from subjects who were enrolled into a site-specific PKsubstudy. For the subjects in the PK substudy, blood samples werecollected before dosing on Day 60, and for a PK profile beginning on Day60 at 30 minutes (corresponding to the end of abatacept infusion), at 4hours after the start of infusion, and weekly thereafter until Day 90. Atotal of 90 subjects were enrolled to participate in the PK substudy.However, complete PK profiles between the dosing interval from Day 60 to90 were obtained from 29 subjects (15 subjects dosed at 2 mg/kg; 14subjects dosed at 10 mg/kg).

A summary of the PK parameters is presented in Table 31. The resultsfrom the study showed that both Cmax and AUC(TAU), where TAU=30 days,increased in a dose proportional manner. For nominal doses increasing inthe ratio of 1:5, the geometric means of Cmax increased in the ratio of1:5.2, while the geometric mean for AUC(TAU) increased in the ratio of1:5.0. In addition, T-HALF, CLT, and Vss values appeared to beindependent of dose. In these RA subjects, the mean T-HALF, CLT, and Vssvalues were around 13 days, ˜0.2 mL/h/kg, and ˜0.07 L/kg, respectively.The small Vss indicates that abatacept is confined primarily to theextracellular fluid volume. Based on the dosing schema of dosing at 2and 4 weeks after the first infusion, then once a month thereafter,steady-state conditions for abatacept were reached by the third monthlydose. Also, as a result of the dosing schema, serum concentrations wereabove steady-state trough concentrations during the first 2 months oftreatment. Comparison of the trough (Cmin) values at Days 60, 90, and180 indicated that abatacept does not appear to accumulate followingmonthly dosing. The mean Cmin steady-state values for all subjectsreceiving monthly IV doses of 2 and 10 mg/kg abatacept ranged between4.4 to 6.7 mcg/mL and 22.0 to 28.7 mcg/mL, respectively.

TABLE 31 Summary of Multiple PK studies in Rheumatoid Arthritis SubjectsProduct Pharmacokinetic Parameters of Abatacept Study ID # Age:Treatment Geometric Mean; (% CV) Mean (SD) Protocol (Batch/ Study StudySubjects (Mean, Dose Cmax AUC (TAU) T-HALF CLT Vss (Country) Lot #)Objective Design (M/Fem) range) (mg/kg) (μg/mL) (μg · h/mL) (Days)(mL/h/kg) (L/kg) IM101100 C00157 Assess the Randomized, 29 54  2.0  54.9 9573.5 13.5 0.23 0.07 (USA) C00196 efficacy, double-blind, (18/11)(34-83) (N = 15) (29) (30) (5.9) (0.13) (0.04) PK C98283 safety,placebo- 10.0 284.2 47624.2 13.1 0.22 0.07 Substudy multiple controlled,(N = 14) (23) (31) (5.3) (0.09) (0.03) Phase II dose PK and multipleimmunogenic dose study, potential of 30-minute IV intravenously infusionadministered doses of abatacept

The pharmacokinetics of abatacept were studied in healthy adult subjectsafter a single 10 mg/kg intravenous infusion and in RA patients aftermultiple 10 mg/kg intravenous infusions (see Table 32).

TABLE 32 Pharmacokinetic Parameters (Mean, Range) in Healthy Subjectsand RA Patients After 10 mg/kg Intravenous Infusion(s) Healthy SubjectsRA Patients (After 10 mg/kg (After 10 mg/kg Single Dose) MultipleDoses^(a)) PK Parameter n = 13 n = 14 Peak Concentration (C_(max))  292(175-427)  295 (171-398) [mcg/mL] Terminal half-life (t_(1/2)) [days]16.7 (12-23) 13.1 (8-25) Systemic clearance (CL) [mL/h/kg] 0.23(0.16-0.30) 0.22 (0.13-0.47) Volume of distribution (Vss) 0.09(0.06-0.13) 0.07 (0.02-0.13) [L/kg] ^(a)Multiple intravenous infusionswere administered at days 1, 15, 30, and monthly thereafter.

The pharmacokinetics of abatacept in RA patients and healthy subjectsappeared to be comparable. In RA patients, after multiple intravenousinfusions, the pharmacokinetics of abatacept showed proportionalincreases of C_(max) and AUC over the dose range of 2 mg/kg to 10 mg/kg.At 10 mg/kg, serum concentration appeared to reach a steady-state by day60 with a mean (range) trough concentration of 24 (1-66) mcg/mL. Nosystemic accumulation of abatacept occurred upon continued repeatedtreatment with 10 mg/kg at monthly intervals in RA patients.

Population pharmacokinetic analyses in RA patients revealed that therewas a trend toward higher clearance of abatacept with increasing bodyweight. Age and gender (when corrected for body weight) did not affectclearance. Concomitant methotrexate (MTX), nonsteroidalanti-inflammatory drugs (NSAIDs), corticosteroids, and TNF blockingagents did not influence abatacept clearance.

Serum Assay for Abatacept

Serum samples were analyzed for abatacept by an enzyme-linkedimmunosorbent assay (ELISA) in a total of 25 analytical runs. Allanalytical results met the acceptance criteria established prior tosample analysis indicating that the ELISA method was precise andaccurate for the quantitation of abatacept in study samples. A summaryof the standard curve parameters and mean QC data for abatacept in serumare presented in Table 33. The between- and within-run variability ofthe analytical QCs for abatacept was 4.5% and 3.5% CV, respectively.Mean observed concentrations of the analytical QC samples deviated lessthan ±8.9% from the nominal values (Table 33).

TABLE 33 Summary of Quality Control Data for the Assay of Abatacept inHuman Serum Low Mid High Nominal Conc. (3.000 ng/mL) (12.500 ng/mL)(24.000 ng/mL) Mean Observed Conc. 2.866 13.608 24.526 % Dev −4.5 8.92.2 Between Run 4.5 2.8 3.0 Precision (% CV) Within Run Precision 2.43.5 2.9 (% CV) Total Variation 5.1 4.5 4.2 (% CV) n 75 75 75 Number ofRuns 25 25 25

Example XI

The objectives of this study are to assess the PK of belataceptfollowing a single SC dose in the range of 50 to 150 mg in healthysubjects; to assess the effects of the injection volume andconcentration of the injected solution on the PK of subcutaneouslyadministered belatacept; to assess the safety and tolerability(including the site of injection evaluation) of a single SC dose ofbelatacept; to assess the immunogenicity of subcutaneously administeredbelatacept.

This is a double-blind, randomized, placebo-controlled, parallel group,single-dose study in healthy subjects. A total of 42 subjects will berandomized to one of 6 treatment groups. Within each group of 7subjects, subjects will be randomized in a 5:2 ratio on Day 1 to receivea single, SC injection of belatacept or placebo. Subjects will berequired to weigh 100 kg. The 6 treatment groups are described in Table34.

TABLE 34 Treatment Groups Treatment Dose of belatacept or InjectinConcentration of the Group Placebo Volume Injected Solution 1 50 mg 0.4mL 125 mg/mL 2 75 mg 0.6 mL 125 mg/mL 3 100 mg  0.8 mL 125 mg/mL 4 150mg  1.2 mL 125 mg/mL 5 50 mg 1.0 mL  50 mg/mL 6 75 mg 1.0 mL  75 mg/mL

Subjects will undergo screening evaluations to determine eligibilitywithin 28 days of dosing on Day 1. Subjects will be admitted to theclinical facility the day prior to dosing (Day −1) for baselineevaluations, including MLR. Subjects will remain in the clinicalfacility until completion of post-treatment assessments on Day 5, andwill return to the clinical facility for each study visit thereafteruntil discharged from the study.

On Day 1 subjects will be randomized to treatment and will receive asingle SC dose of belatacept or placebo, and undergo detailed PK andimmunogenicity sampling. All subjects will receive the SC injections intheir anterior thigh. Following study drug administration, theInvestigator will assess the injection site for signs of localirritation and inflammation.

Physical examinations, vital sign measurements, and clinical laboratoryevaluations will be performed at selected times throughout the study.Blood samples will be collected for up to 56 days after study drugadministration for PK analysis and assessment of immunogenicity.Subjects will be monitored for AEs throughout the study. Approximately265 mL of blood will be drawn from each subject during the study.

Dosing and follow-up will occur concurrently for all dose groups. Nosubject will receive more than a single dose. Subjects who do notcomplete the study (except those who are discontinued for AEs) may bereplaced.

This is a single dose study. Each subject will undergo a screeningperiod which will be a maximum of 28 days prior to the day study drug isadministered. Each subject will remain in the study until the lastvisit, 56 Days (±2 Days) after study drug is administered. The lastvisit of the last subject undergoing the trial will be considered theend of the study.

Belatacept 100 mg/vial (125 mg/mL), as described herein, andmanufactured as described in co-pending U.S. patent application Ser. No.60/849,543, filled on Oct. 5, 2006 which teaches processes for theproduction of proteins of the invention, specifically recombinantglycoprotein products, by animal or mammalian cell cultures, is aready-to-use liquid product provided in a glass vial for withdrawal andadministration using a suitable size conventional syringe and needle forSC administration. A sufficient excess of belatacept is incorporatedinto each vial to account for withdrawal losses so that 0.8 mL of thesolution containing 100 mg can be withdrawn for SC administration.

Belatacept Injection, 100 mg/vial (125 mg/mL) is not intended for an IVinfusion

Healthy subjects as determined by medical history, physical examination,12-lead electrocardiogram, and clinical laboratory evaluations will beeligible to participate in the study. This study will include men andwomen. Subjects must be at least 18 years of age and weigh 100 kg at thetime of randomization. Female subjects must be not nursing, not pregnantand must be using an acceptable method of contraception for at least 1month before dosing, during the study and for up to 4 weeks after theend of the study. Women of childbearing potential must have a negativeserum pregnancy test within 24 hours prior to the dose of studymedication. Subjects will be advised on potential risks to a pregnancy.Male subjects must be using an adequate method of contraception duringthe study and for up to 4 weeks after the end of the study so that therisk of pregnancy to their partner is minimized. See Section 5 for adetailed list of the inclusion and exclusion criteria.

Medications taken within 4 weeks prior to enrollment must be recorded onthe CRF. No concomitant medications (prescription, over-the-counter orherbal) are to be administered during study, except for oralcontraceptives, unless they are prescribed by the Investigator fortreatment of specific clinical events. Any concomitant therapies must berecorded on the CRF.

PK of belatacept following SC injection will be derived from serumconcentration versus time data. The single-dose PK parameters to beassessed include:

-   Cmax Maximum observed serum concentration-   Tmax Time of maximum observed serum concentration-   AUC(0-T) Area under the serum concentration-time curve from time    zero to the time of the last quantifiable concentration-   AUC(INF) Area under the serum concentration-time curve from time    zero extrapolated to infinite time-   Half life Serum half-life-   CLT/F Apparent total body clearance-   VSS/F Apparent volume of distribution at steady state

Individual subject PK parameter values will be derived bynon-compartmental methods by a validated PK analysis program, theeToolbox Kinetica program, Innaphase Corp, Philadelphia, Pa. Dosenormalized AUC will also be reported.

Serum samples will be collected over time and assayed for the presenceof antibody titers to belatacept using two ELISA assays. One assayassesses the response to the whole molecule and the other to theLEA29Y-T portion only.

All subjects who receive study medication will be included in the safetyand PD data sets. Subjects who receive placebo in any panel will bepooled into a single placebo treatment group for PD assessments andsafety assessments except for the site of injection assessments. Allavailable data from subjects who receive belatacept will be included inthe PK data set, and will be included in the summary statistics andstatistical analysis.

Baseline is considered Day −1. Frequency distributions of gender andrace will be tabulated by treatment (injection volume and dose). Summarystatistics for age, body weight, and height will be tabulated bytreatment.

All recorded AEs will be listed and tabulated by preferred term, systemorgan class, and treatment. Vital signs and clinical laboratory testresults will be listed and summarized by treatment. Any significantphysical examination findings and clinical laboratory results will belisted. Injection site assessments (erythema, heat, swelling, pain andpruritus) will be tabulated by treatment and degree of severity. Placebosubjects will be pooled across dose groups and analyzed independently,as well, for the assessment of site of injection.

Summary statistics will be tabulated for the PK parameters by treatment.Geometric means and coefficients of variation will be presented forCmax, AUC(0-T), and AUC(INF). Medians, minima, and maxima will bepresented for Tmax. Means and standard deviations will be provided forother PK parameters. To assess the dependency on dose after SCadministration, scatter plots of Cmax and AUC(INF) versus dose will beprovided. Scatter plots of AUC(INF) and Cmax across injection volumes,will be constructed to assess this effect on the PK of belatacept. Also,scatter plots of Cmax and AUC(INF) versus dose for a fixed volume, andCmax and AUC(INF) versus volume within doses will be provided whereapplicable.

Summary statistics will be tabulated by treatment and study day foranti-belatacept and anti-LEA29Y-T antibody values and their changes frombaseline (Day 1-0 hr). To explore possible associations betweenimmunogenicity and exposure, plots of changes in anti-belatacept andanti-LEA29Y-T antibodies versus belatacept concentrations will beprovided.

PK and immunogenicity blood sampling schedules are outlined in Table 35.

TABLE 35 Pharmacokinetic and Immunogenicity Blood Sampling Schedule Time(Relative to Dosing) Study Day hours:min PK Immunogenicity 1 00.00(predose) X X^(a) 01:00 X 02:00 X 06:00 X 12:00 X 2 24:00 X 36:00 X 348:00 X 60:00 X 4 72:00 X 84:00 X 5 96:00 X 6 120:00  X 7 144:00  X 8168:00  X 14 X X 21 X 28 X X 35 X X 42 X X 56 X X ^(a)Day 1immunogenicity (anti-belatacept antibodies) sample must be drawn priorto administering drug. Samples on Days 28, 35, 42 and 56 can be drawnwithin ±2 days.

Table 35 above lists the sampling schedule to be followed for theassessment of PK. Blood samples (˜3 mL per sample) will be collectedinto a pre-labeled, red and gray top (SST) Vacutainer® tube via directvenipuncture or from a saline lock. If a saline lock system is used forblood collection, approximately 0.5 mL of blood should be withdrawnthrough the indwelling catheter and be discarded prior to obtaining eachPK sample. Once the PK specimen has been obtained, the blood will beallowed to clot in the Vacutainer® tube at room temperature for 15-30minutes. Following clotting, the sample must be centrifuged for 15minutes at 1500×g in a refrigerated centrifuge (4° C.). Whencentrifugation is complete, at least 0.5 mL of serum from each PK sampletime point should be removed by pipette and transferred to a prelabeled,screw cap, polypropylene, PK storage and shipping tube. A clean pipettemust be used to aliquot serum for each sample time point. Thepolypropylene tube containing the PK serum sample may be stored frozenat −20° C. or colder for a maximum of one month and then at −70° C.,thereafter. The time permitted from sample collection to freezing of theserum is 12 hours. A sensitive, validated enzyme immunoassay (EIA)method will be used to measure concentrations of belatacept in serum.

Table 35 lists the sampling schedule to be followed for the assessmentof immunogenicity. Serum samples will be obtained at visits Days 1, 14,28, 35, 42, and 56. The Day 1 immunogenicity (anti-belatacept) sampleshould be taken prior to administering study drug. Samples will beassayed for the presence of anti-belatacept and anti-LEA29Y-Tantibodies. For each specimen, blood (˜3 mL per sample) will becollected into a pre-labeled red and gray top (SST) Vacutainer® tube viadirect venipuncture or from a saline lock (indwelling catheter). If asaline lock system is used for blood collection, approximately 0.5 mL ofblood should be drawn through the indwelling catheter and be discardedprior to obtaining each immunogenicity sample. Once the specimen hasbeen obtained, the blood will be allowed to clot in the Vacutainer® tubeat room temperature for 15-30 minutes. Following clotting, the samplemust be centrifuged for 15 minutes at 1500×g in a refrigeratedcentrifuge (4° C.). When centrifugation is complete, at least 1 mL ofserum should be removed by pipette and transferred to a prelabeled,screw cap, polypropylene, serum sample storage and shipping tube. Thepolypropylene tube containing the serum sample must be stored frozen at−20° C. or colder. Two sensitive, validated enzyme linked immunosorbentassay (ELISA) methods will be used to measure antibody titers tobelatacept in serum. One assay assess the antibody titer to the wholemolecule and the other to the LEA29Y-T portion only.

Example XII

Deamidation, fragmentation and aggregation are observed degradationpathways of LEA29YIg molecules. This protocol outlines an additionallaboratory scale pH stability study designed to evaluate the belataceptSC drug product formulation in the pH range of 6.3-7.5. The purpose ofthis study is to identify the optimal pH formulation that will attain aminimum of 18-months of shelf-life for the belatacept SC formulation.

For this study, belatacept SC product will be formulated at pH 6.3, 6.6,6.9, 7.2 and 7.5. The belatacept drug substance at ˜25 mg/mL will befirst concentrated to ˜100 mg/mL, then diafiltered into 10 mM phosphatebuffer at pH 6.9 followed by second concentration to obtain a drugproduct intermediate (DPI) at >160 mg/mL. The DPI will be formulatedwith sucrose and poloxamer 188 and the final batch concentration will beadjusted with 10 mM phosphate buffer (pH 6.9). The formulated bulk willbe subdivided into five sub-batches, as outlined in the study design.The pH of the sub-batches will be titrated down to 6.3 and 6.6,respectively, using 1N HCl. The pH of the additional two sub-batcheswill be titrated up to 6.9, 7.2 and 7.5 with 1N NaOH. The productbatches will be filled into 1-mL long Physiolis™. Samples should beprotected from light at all times by covering or inserting into brownlight-protective bags. The SC drug product formulation utilized in thisstudy is described in Table 36 below.

TABLE 36 SC drug product formulation composition at pH 6.9 Amount per1.0 mL Ingredient (mg) Belatacept 125 Sucrose 170 Poloxamer 188 8Monobasic sodium phosphate, monohydrate 0.638 Dibasic sodium phosphate,anhydrous 0.475 Water for Injection QS 1.0 mL

Drug product stored at 2-8° C. will be sampled at 0, 2, 4, 6, 12 18, 24months and optionally 9 months. Drug product stored at 15° C. will besampled at 1, 2, 4, 6 months and optionally 9 months. Drug productstored at 25° C. and 60% humidity will be sampled at 1, 2, 4 and 6months. Drug product stored at 35° C. will be sampled at 1, 2 and 4months. Samples stored at 2-8° C. will be tested for appearance (initialand last samples only), pH (initial, 4 month and last samples only),A280(initial, 4 month and last samples only), size exclusion-HPLC,SDS-PAGE, tryptic digest peptide mapping (TPM), Biacore B7 Binding(initial, 4 month and last samples only or as needed) and isoelectricfocusing(IEF)(initial and last samples only). Samples stored at 15° C.and 25° C. with 60% humidity will be tested for A280(initial, 4 monthsand last samples only), size exclusion-HPLC, SDS-PAGE and tryptic digestpeptide mapping (TPM). Samples stored at 35° C. will be tested for sizeexclusion-HPLC, SDA-PAGE and tryptic digest peptide mapping (TPM).

Non-Routine testing methods will be used to further characterizestability samples at the initial time point, 4 months, 12 months and atthe end of the study. Some of these methods may also be used to testspecific samples if a trend or an unexpected result is observed. Thenon-routine methods include: size exclusion chromatography employingmultiangle light scattering (SEC-MALS), kinetic binding (SPR), MassSpectrometry, CD, AUC, Differential Scanning calorimeter (DSC), FFF,FTIR, size exclusion-HPLC (denatured) and SDS-PAGE (silver stain).

What is claimed:
 1. A method for treating rheumatoid arthritiscomprising administering to a subject in need thereof an effectiveamount of a stable formulation suitable for subcutaneous administrationcomprising the CTLA4Ig molecule having the amino acid sequence shown inSEQ ID NO:2 starting at methionine at position 27 or alanine at position26 and ending at lysine at position 383 or glycine at position 382 in anamount of about 125 mg/ml, sucrose in an amount of about 170 mg/ml, atleast one buffering agent, sterile water for injection and a surfactant,wherein the formulation has a pH range of from 6 to 7.8, and wherein theformulation is administered in a 1 ml volume.
 2. A method for treatingrheumatoid arthritis comprising administering to a subject in needthereof an effective amount of a stable formulation suitable forsubcutaneous administration comprising the CTLA4Ig molecule having theamino acid sequence shown in SEQ ID NO:2 starting at methionine atposition 27 or alanine at position 26 and ending at lysine at position383 or glycine at position 382 in an amount of about 125 mg/ml, sucrosein an amount of about 170 mg/ml, at least one buffering agent, sterilewater for injection and a surfactant, wherein the formulation has a pHrange of from 6 to 7.8, and wherein the formulation is administered oncea week in a 1 ml volume.
 3. The method of claim 1 or 2 wherein thestable formulation suitable for subcutaneous administration is stablewhen stored at 2 to 8° C. for at least 12 months.
 4. The method of claim1 or 2 wherein the stable formulation suitable for subcutaneousadministration has an osmolality of from 700 mOsm/kgH2O to 800mOsm/kgH₂O.
 5. The method of claim 1 or 2 wherein the stable formulationsuitable for subcutaneous administration has a viscosity of from 9 to 20mPa·s
 6. The method of claim 1 or 2, wherein the surfactant is selectedfrom the group consisting of polysorbates, polysorbate 20, polysorbate80, poloxamers, poloxamer 188, sorbitan esters, sorbitan derivatives,Triton, sodium laurel sulfate, sodium octyl glycoside,lauryl-sulfobetadine, myristyl-sulfobetadine, linoleyl-sulfobetadine,stearyl-sulfobetadine, lauryl-sarcosine, myristyl-sarcosine,linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine,myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine,cocamidopropyl-betaine, linoleamidopropyl-betaine,myristamidopropyl-betaine, palmidopropyl-betaine,isostearamidopropyl-betaine, lauroamidopropyl-dimethylamine,myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine,isostearamidopropyl-dimethylamine, sodium methyl cocoyl-taurate, anddisodium methyl oleyl-tauratem, polyethylene glycol, polypropyl glycol,and copolymers of ethylene and propylene glycol.
 7. The method of claim6, wherein the surfactant is poloxamer
 188. 8. The method of claim 1 or2, wherein the buffering agent is selected from the group consisting ofdibasic sodium phosphate anhydrous, monobasic sodium phosphatemonohydrate, glycinate buffers, carbonate buffers, citrate buffers andcombinations thereof.
 9. The method of claim 8 wherein the bufferingagent is in an amount of at least 10 mM phosphate buffer.
 10. The methodof claim 1 or 2, wherein the stable formulation suitable forsubcutaneous administration further comprises a preservative.
 11. Themethod of claim 10, wherein the preservative is selected from the groupconsisting of octadecyldimethylbenzyl ammonium chloride, hexamethoniumchloride, benzalkonium chloride, benzethonium chloride, phenol, butylalcolhol, benzyl alcohol, alkyl parabens, methyl paraben, propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.12. The method of claim 3, wherein the percentage of CTLA4Ig highmolecular weight species in the stable formulation suitable forsubcutaneous administration is less than about 25%.
 13. The method ofclaim 3, wherein the percentage of CTLA4Ig high molecular weight speciesin the stable formulation suitable for subcutaneous administration isless than about 15%.
 14. The method of claim 3, wherein the percentageof CTLA4Ig high molecular weight species in the stable formulationsuitable for subcutaneous administration is less than about 10%.
 15. Themethod of claim 3, wherein the percentage of CTLA4Ig high molecularweight species in the stable formulation suitable for subcutaneousadministration is less than about 5%.
 16. The method of claim 3, whereinthe percentage of CTLA4Ig high molecular weight species in the stableformulation suitable for subcutaneous administration is less than about3%.